Optimized production and purification of Bacillus anthracis lethal factor.

Bacillus anthracis lethal factor (LF) is a 90-kDa zinc metalloprotease that plays an important role in the virulence of the organism. LF has previously been purified from Escherichia coli and Bacillus anthracis. The yields and purities of these preparations were inadequate for crystal structure determination.

Clostridium septicum alpha toxin uses glycosylphosphatidylinositol-anchored protein receptors.

The alpha toxin produced by Clostridium septicum is a channel-forming protein that is an important contributor to the virulence of the organism. Chinese hamster ovary (CHO) cells are sensitive to low concentrations of the toxin, indicating that they contain toxin receptors. Using retroviral mutagenesis, a mutant CHO line (BAG15) was generated that is resistant to alpha toxin.

Anthrax toxin fusion proteins for intracellular delivery of macromolecules.

The dominant role played by the anthrax toxin in Bacillus anthracis pathogenesis shows that the toxin has evolved to be an efficient system for delivering its two catalytic protein components, oedema factor and lethal factor (LF), into the cytosol of host cells. This system involves binding of the protective antigen (PA) toxin component to a ubiquitous (and still unidentified) receptor, proteolytic activation at the cell surface, internalization by endocytosis and translocation through an early endosome membrane to the cytosol (Leppla 1995). We and colleagues showed that the system can be exploited to deliver heterologous polypeptides to the cytosol (Arora et al.

Crystallographic studies of the anthrax lethal toxin.

Anthrax lethal toxin comprises two proteins: protective antigen (PA; MW 83 kDa) and lethal factor (LF; MW 87 kDa). We have recently determined the crystal structure of the 735-residue PA in its monomeric and heptameric forms (Petosa et al. 1997).

Proteasome activity is required for anthrax lethal toxin to kill macrophages.

Anthrax lethal toxin (LeTx), consisting of protective antigen (PA) and lethal factor (LF), rapidly kills primary mouse macrophages and macrophage-like cell lines such as RAW 264.7. LF is translocated by PA into the cytosol of target cells, where it acts as a metalloprotease to cleave mitogen-activated protein kinase kinase 1 (MEK1) and possibly other proteins.

Identification of a receptor-binding region within domain 4 of the protective antigen component of anthrax toxin.

Anthrax toxin from Bacillus anthracis is a three-component toxin consisting of lethal factor (LF), edema factor (EF), and protective antigen (PA). LF and EF are the catalytic components of the toxin, whereas PA is the receptor-binding component. To identify residues of PA that are involved in interaction with the cellular receptor, two solvent-exposed loops of domain 4 of PA (amino acids [aa] 679 to 693 and 704 to 723) were mutagenized, and the altered proteins purified and tested for toxicity in the presence of LF.

Oligomerization of anthrax toxin protective antigen and binding of lethal factor during endocytic uptake into mammalian cells.

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.

Protection against anthrax toxin by vaccination with a DNA plasmid encoding anthrax protective antigen.

A DNA vaccine encoding the immunogenic and biologically active portion of anthrax protective antigen (PA) was constructed. Spleen cells from BALB/c mice immunized intramuscularly with this vaccine were stimulated to secrete IFN gamma and IL-4 when exposed to PA in vitro. Immunized mice also mounted a humoral immune response dominated by IgG1 anti-PA antibody production, the subclass previously shown to confer protection against anthrax toxin.

The pore-forming toxin proaerolysin is activated by furin.

Aerolysin is secreted as an inactive dimeric precursor by the bacterium Aeromonas hydrophila. Proteolytic cleavage within a mobile loop near the C terminus of the protoxin is required for oligomerization and channel formation. This loop contains the sequence KVRRAR432, which should be recognized by mammalian proprotein convertases such as furin, PACE4, and PC5/6A.

Study of immunization against anthrax with the purified recombinant protective antigen of Bacillus anthracis.

Protective antigen (PA) of anthrax toxin is the major component of human anthrax vaccine. Currently available human vaccines in the United States and Europe consist of alum-precipitated supernatant material from cultures of toxigenic, nonencapsulated strains of Bacillus anthracis. Immunization with these vaccines requires several boosters and occasionally causes local pain and edema.

Proteolytic inactivation of MAP-kinase-kinase by anthrax lethal factor.

Anthrax lethal toxin, produced by the bacterium Bacillus anthracis, is the major cause of death in animals infected with anthrax. One component of this toxin, lethal factor (LF), is suspected to be a metalloprotease, but no physiological substrates have been identified. Here it is shown that LF is a protease that cleaves the amino terminus of mitogen-activated protein kinase kinases 1 and 2 (MAPKK1 and MAPKK2) and that this cleavage inactivates MAPKK1 and inhibits the MAPK signal transduction pathway.

Internalization of a Bacillus anthracis protective antigen-c-Myc fusion protein mediated by cell surface anti-c-Myc antibodies.

Background: Anthrax toxin, secreted by Bacillus anthracis, consists of protective antigen (PA) and either lethal factor (LF) or edema factor (EF). PA, the receptor-binding component of the toxin, translocates LF or EF into the cytosol, where the latter proteins exert their toxic effects. We hypothesized that anthrax toxin fusion proteins could be used to kill virus-infected cells and tumor cells, if PA could be redirected to unique receptors found only on these cells.

Targeting HIV proteins to the major histocompatibility complex class I processing pathway with a novel gp120-anthrax toxin fusion protein.

A challenge for subunit vaccines whose goal is to elicit CD8(+) cytotoxic T lymphocytes (CTLs) is to deliver the antigen to the cytosol of the living cell, where it can be processed for presentation by major histocompatibility complex (MHC) class I molecules. Several bacterial toxins have evolved to efficiently deliver catalytic protein moieties to the cytosol of eukaryotic cells. Anthrax lethal toxin consists of two distinct proteins that combine to form the active toxin.

Clostridium septicum alpha-toxin is proteolytically activated by furin.

Clostridium septicum alpha-toxin is secreted as an inactive 46,450-Da protoxin. The protoxin is activated by proteolytic cleavage near the C terminus, which eventually causes the release of a 45-amino-acid fragment. Proteoytic activation and loss of the propeptide allow alpha-toxin to oligomerize and form pores on the plasma membrane, which results in colloidal-osmotic lysis.

A role for PACE4 in the proteolytic activation of anthrax toxin protective antigen.

Several bacterial protein toxins require activation by eukaryotic proteases. Previous studies have shown that anthrax toxin protective antigen (PA), Pseudomonas exotoxin A (PE), and diphtheria toxin (DT) are cleaved by furin C-terminal to the sequences RKKR, RQPR, and RVRR, respectively. Because furin-deficient cells retain some sensitivity to PA and DT, it is evident that other cellular proteases can activate these toxins.

Delivery of antigens to the MHC class I pathway using bacterial toxins.

Cytotoxic T lymphocytes (CTL) recognize antigens derived from endogenously expressed proteins presented on the cell surface in the context of major histocompatibility complex (MHC) class I molecules. Because CTL are effective in antiviral and antitumor responses, the delivery of antigens to the class I pathway has been the focus of numerous efforts. Generating CTL by immunization with exogenous proteins is often ineffective because these antigens typically enter the MHC class II pathway.

Crystal structure of the anthrax toxin protective antigen.

Protective antigen (PA) is the central component of the three-part protein toxin secreted by Bacillus anthracis, the organism responsible for anthrax. After proteolytic activation on the host cell surface, PA forms a membrane-inserting heptamer that translocates the toxic enzymes, oedema factor and lethal factor, into the cytosol. PA, which has a relative molecular mass of 83,000 (M(r) 83K), can also translocate heterologous proteins, and is being evaluated for use as a general protein delivery system.

Characterization of lethal factor binding and cell receptor binding domains of protective antigen of Bacillus anthracis using monoclonal antibodies.

Lethal toxin from Bacillus anthracis is composed of protective antigen (PA) and lethal factor (LF). Anti-PA mAbs that neutralized lethal toxin activity, either in vivo or in vitro, identified three non-overlapping antigenic regions on PA. Two distinct antigenic regions were recognized by the four mAbs that neutralized lethal toxin activity by inhibiting the binding of 125I-LF to cell-bound PA.

Furin regulates both the activation of Pseudomonas exotoxin A and the Quantity of the toxin receptor expressed on target cells.

Pseudomonas exotoxin A (PE) binds and enters mammalian cells via the alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (LRP). The toxin then requires proteolytic cleavage to generate an enzymatically active fragment with translocates to the cell cytosol and inhibits protein synthesis. To assess the role of furin in determining toxin susceptibility, CHO cells were transfected with a mouse furin gene (CHO+fur cells) and maintained under neomycin selection.

Pseudomonas exotoxin-mediated selection yields cells with altered expression of low-density lipoprotein receptor-related protein.

The alpha 2-macroglobulin (alpha 2M) receptor/low-density lipoprotein receptor-related protein (LRP) is important for the clearance of proteases, protease-inhibitor complexes, and various ligands associated with lipid metabolism. While the regulation of receptor function is poorly understood, the addition of high concentrations of the 39-kD receptor-associated protein (RAP) to cells inhibits the binding and/or uptake of many of these ligands. Previously, we (Kounnas, M.

Furin is important but not essential for the proteolytic maturation of gp160 of HIV-1.

The envelope glycoproteins of HIV are required for viral infectivity. Proteolysis of the precursor envelope glycoprotein gp160 results in the formation of gp120 and gp41. Cleavage occurs after the sequence Arg-Glu-Lys-Arg.

In vitro processing of anthrax toxin protective antigen by recombinant PC1 (SPC3) and bovine intermediate lobe secretory vesicle membranes.

Protective antigen (PA), an 83-kDa protein produced by Bacillus anthracis, requires proteolytic activation at a tetrabasic site (RKKR167) before it can combine with either edema factor or lethal factor on the cell surface. The complex is then endocytosed and the target cell intoxicated. Previous work has demonstrated that furin, a ubiquitously distributed, subtilisin-like protease, can perform this cleavage.

Proteolytic activation of bacterial toxins by eukaryotic cells is performed by furin and by additional cellular proteases.

Before intoxication can occur, anthrax toxin protective antigen (PA), Pseudomonas exotoxin A (PE), and diphtheria toxin (DT) must be activated by proteolytic cleavage at specific amino acid sequences. Previously, it was shown that PA and DT can be activated by furin. In Chinese hamster ovary (CHO) cells, wild-type (RKKR) and cleavage site mutants of PA, each administered with a modified form of anthrax toxin lethal factor (the N terminus of lethal factor fused to PE domain III), had the following potencies: RKKR (wild type) (concentration causing 50% cell death [EC50] = 12 ng/ml) > or = RAAR (EC50 = 18 ng/ml) > FTKR (EC50 = 24 ng/ml) > STRR (EC50 = 49 ng/ml).

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

The protective antigen (PA) component of anthrax toxin contains two sites that are uniquely sensitive to proteolytic cleavage. Cleavage at the sequence RKKR167 by the cellular protease furin is absolutely required for toxicity, whereas cleavage by chymotrypsin or thermolysin at the sequence FFD315 inactivates the protein, apparently by blocking the ability of PA to translocate the catalytic moieties of the toxins, lethal factor (LF) and edema factor (EF), to the cytosol of eukaryotic cells. To specify the role of the chymotrypsin-sensitive site of PA in the translocation of LF, we altered residues 313-315.

Fusions of anthrax toxin lethal factor with shiga toxin and diphtheria toxin enzymatic domains are toxic to mammalian cells.

To investigate the ability of anthrax toxin lethal factor (LF) to translocate foreign proteins into the cytosol of eukaryotic cells and to characterize the structural requirements of this process, fusion proteins containing a portion of LF and the catalytic domains of either diphtheria toxin or Shiga toxin were constructed. Previous work showed that residues 1 to 254 of anthrax toxin lethal factor (LF1-254) are sufficient for binding to the protective antigen component of the toxin and that portions of Pseudomonas exotoxin A fused to LF1-254 are efficiently translocated to the cytosol of eukaryotic cells (N. Arora and S.