Optimized Production and Purification of Bacillus anthracis Lethal Factor

Park, Sukjoon; Leppla, Stephen H.

doi:10.1006/prep.2000.1208

Cited by 115 publications

(110 citation statements)

References 35 publications

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“…Recombinant Bacillus anthracis LF and PA were produced as described in ref. 21 or acquired from List Biological Laboratories (Campbell, CA). ␤-Cyclodextrin and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma, and AmPr␤CD was custom synthesized at Pinnacle Pharmaceuticals (Charlottesville, VA).…”

Section: Methodsmentioning

confidence: 99%

Blocking anthrax lethal toxin at the protective antigen channel by using structure-inspired drug design

Karginov

Nestorovich

Moayeri

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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111

160

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Bacillus anthracis secretes three polypeptides: protective antigen (PA), lethal factor (LF), and edema factor (EF), which interact at the surface of mammalian cells to form toxic complexes. LF and EF are enzymes that target substrates within the cytosol; PA provides a heptameric pore to facilitate LF and EF transport into the cytosol. Other than administration of antibiotics shortly after exposure, there is currently no approved effective treatment for inhalational anthrax. Here we demonstrate an approach to disabling the toxin: high-affinity blockage of the PA pore by a rationally designed low-molecular weight compound that prevents LF and EF entry into cells. Guided by the sevenfold symmetry and predominantly negative charge of the PA pore, we synthesized small cyclic molecules of sevenfold symmetry, ␤-cyclodextrins chemically modified to add seven positive charges. By channel reconstitution and high-resolution conductance recording, we show that per-6-(3-aminopropylthio)-␤-cyclodextrin interacts strongly with the PA pore lumen, blocking PA-induced transport at subnanomolar concentrations (in 0.1 M KCl). The compound protected RAW 264.7 mouse macrophages from cytotoxicity of anthrax lethal toxin ‫؍(‬ PA ؉ LF). More importantly, it completely protected the highly susceptible Fischer F344 rats from lethal toxin. We anticipate that this approach will serve as the basis for a structure-directed drug discovery program to find new and effective treatments for anthrax.infectious diseases ͉ membrane transport ͉ modified cyclodextrins

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Section: Methodsmentioning

confidence: 99%

Blocking anthrax lethal toxin at the protective antigen channel by using structure-inspired drug design

Karginov

Nestorovich

Moayeri

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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111

160

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“…Synthetic LL-37 was generously provided by Hubert Kalbacher (University of Tübingen, Tü-bingen, Germany). Recombinant LF and PA were purchased from Calbiochem or purified from recombinant B. anthracis strains kindly provided by Stephen H. Leppla (National Institute of Allergy and Infectious Diseases, Bethesda) (15,16).…”

Section: Methodsmentioning

confidence: 99%

Human α-defensins neutralize anthrax lethal toxin and protect against its fatal consequences

Kim

Gajendran

Mittrücker

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

143

125

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Anthrax caused by Bacillus anthracis represents a major bioterroristic threat. B. anthracis produces lethal toxin (LeTx), a combination of lethal factor (LF) and protective antigen that plays a major role in anthrax pathogenesis. We demonstrate that human neutrophil ␣-defensins are potent inhibitors of LF. The inhibition of LF by human neutrophil protein (HNP-1) was noncompetitive. HNP-1 inhibited cleavage of a mitogen-activated protein kinase kinase and restored impaired mitogen-activated protein kinase signaling in LeTx-treated macrophages. HNP-1 rescued murine macrophages from B. anthracis-induced cytotoxicity, and in vivo treatment with HNP-1-3 protected mice against the fatal consequences of LeTx.defensin ͉ innate immunity ͉ Bacillus anthracis

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“…Phenyl-Sepharose Fast Flow and Sephacryl S-100 were purchased from Amersham Biosciences. Wild type PA, LF, and FP59 were purified in our laboratory as previously described (29,30). Rabbit anti-PA antibody (number 5308) was made in our laboratory, and goat anti-rabbit IgG-horseradish peroxidase (sc2054) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).…”

Section: Methodsmentioning

confidence: 99%

Alanine-scanning Mutations in Domain 4 of Anthrax Toxin Protective Antigen Reveal Residues Important for Binding to the Cellular Receptor and to a Neutralizing Monoclonal Antibody

Rosovitz

Schuck

Varughese

et al. 2003

Journal of Biological Chemistry

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A panel of variants with alanine substitutions in the small loop of anthrax toxin protective antigen domain 4 was created to determine individual amino acid residues critical for interactions with the cellular receptor and with a neutralizing monoclonal antibody, 14B7. Substituted protective antigen proteins were analyzed by cellular cytotoxicity assays, and their interactions with antibody were measured by plasmon surface resonance and analytical ultracentrifugation. Residue Asp 683 was the most critical for cell binding and toxicity, causing an ϳ1000-fold reduction in toxicity, but was not a large factor for interactions with 14B7. Substitutions in residues Tyr 681 , Asn 682 , and Pro 686 also reduced toxicity significantly, by 10 -100-fold. Of these, only Asn 682 and Pro 686 were also critical for interactions with 14B7. However, residues Lys 684 , Leu 685 , Leu 687 , and Tyr 688 were critical for 14B7 binding without greatly affecting toxicity. The K684A and L685A variants exhibited wild type levels of toxicity in cell culture assays; the L687A and Y688A variants were reduced only 1.5-and 5-fold, respectively.Bacillus anthracis secretes two toxins: edema toxin and lethal toxin. Each is composed of a common binding component, protective antigen (PA), 1 together with an enzymatic component, edema factor (EF), in the case of edema toxin and lethal factor (LF) in the case of lethal toxin (1-3). The current model for toxin entry into the cell illustrates the centrality of PA for toxin action. PA binds to cellular receptors, recently identified as splice variants of either tumor endothelial marker 8 (TEM8) (4 -6) or the closely related capillary morphogenesis protein 2 (CMG2) (7). Furin cleaves PA, releasing a 20-kDa fragment and leaving behind a 63-kDa portion (PA 63 ) capable of forming a heptamer, which has a newly exposed surface that binds . Heptamer complexes enter the endocytic pathway by receptor-mediated endocytosis (13), and upon acidification of the vesicle, the PA 63 heptamer undergoes a conformational change to form a pore through which EF and LF translocate into the cytoplasm (10, 11, 14 -16). Once in the cytoplasm, EF and LF exert their toxic effects.The PA protein can be divided into four domains based on its crystal structure, and functions can be attributed to the different domains based on mutational and biochemical analyses (16). Domain 1 (residues 1-258) contains the furin cleavage site as well as the hydrophobic portion of PA, which is exposed upon furin cleavage to allow EF and LF to bind (16,17). Several lines of evidence indicate that domain 2 (residues 259 -487) is involved in oligomerization and contains the loop that inserts into the membrane to form the channel through which the LF and EF enter the cytosol (16, 18 -20). Various amino acids in domain 3 (residues 488 -595) are necessary for oligomerization, and this has been the only function attributed to domain 3 to date (21,22). Domain 4 (residues 596 -735) is essential for binding to cellular receptor as indicated by several lines of...

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Optimized Production and Purification of Bacillus anthracis Lethal Factor

Cited by 115 publications

References 35 publications

Blocking anthrax lethal toxin at the protective antigen channel by using structure-inspired drug design

Blocking anthrax lethal toxin at the protective antigen channel by using structure-inspired drug design

Human α-defensins neutralize anthrax lethal toxin and protect against its fatal consequences

Alanine-scanning Mutations in Domain 4 of Anthrax Toxin Protective Antigen Reveal Residues Important for Binding to the Cellular Receptor and to a Neutralizing Monoclonal Antibody

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