https://mmbr.asm.org/content/mmbr/56/4/622.full.pdf

Structure and function of cholera toxin and the related Escherichia coli heat-labile enterotoxin.

The bacterium Bacillus anthracis causes the death of macrophages, which may allow it to avoid detection by the innate immune system. We found that B. anthracis lethal factor (LF) selectively induces apoptosis of activated macrophages by cleaving the amino-terminal extension of mitogen-activated protein kinase (MAPK) kinases (MKKs) that activate p38 MAPKs. Because macrophages that are deficient in transcription factor nuclear factor κB (NF-κB) are also sensitive to activation-induced death and p38 is required for expression of certain NF-κB target genes, p38 is probably essential for synergistic induction of those NF-κB target genes that prevent apoptosis of activated macrophages. This dismantling of the p38 MAPK module represents a strategy used by B. anthracis to paralyze host innate immunity.

/pdf/macrophage-apoptosis-by-anthrax-lethal-factor-through-p38-1478a28n68.pdf

Macrophage Apoptosis by Anthrax Lethal Factor Through p38 MAP Kinase Inhibition

Proteolytic cleavage of the protective antigen (PA) protein of anthrax toxin at residues 164-167 is necessary for toxic activity. Cleavage by a cellular protease at this sequence, Arg-Lys-Lys-Arg, normally follows binding of PA to a cell surface receptor. We attempted to identify this protease by determining its sequence specificity and catalytic properties. Semi-random cassette mutagenesis was used to generate mutants with replacements of residues 164-167 by Arg, Lys, Ser, or Asn. Analysis of 19 mutant proteins suggested that lethal factor-dependent toxicity required the sequence Arg-Xaa-Xaa-Arg. Based on these data, three additional mutants were constructed with the sequences Ala-Lys-Lys-Arg, Arg-Lys-Lys-Ala, and Arg-Ala-Ala-Arg. Of these mutant proteins, Arg-Ala-Ala-Arg was toxic, confirming that the cellular protease can recognize the sequence Arg-Xaa-Xaa-Arg. The mutant containing the sequence Ala-Lys-Lys-Arg was also toxic but required > 13 times more protein to produce equivalent toxicity. This sequence specificity is similar to that of the ubiquitous subtilisin-like protease furin, which is involved in processing of precursors of certain receptors and growth factors. Therefore we tested whether a recombinant soluble furin would cleave PA. This furin derivative efficiently cleaved native PA and the Arg-Ala-Ala-Arg mutant but not the nontoxic PA mutants. In addition, previously identified inhibitors of furin blocked cleavage of receptor-bound PA. These data imply that furin is the cellular protease that activates PA, and that nearly all cell types contain at least a small amount of furin exposed on their cell surface.

https://www.pnas.org/content/pnas/89/21/10277.full.pdf

Anthrax toxin protective antigen is activated by a cell surface protease with the sequence specificity and catalytic properties of furin

Certain pathogenic species of Bacillus and Clostridium have developed unique methods for intoxicating cells that employ the classic enzymatic “A-B” paradigm for protein toxins. The binary toxins produced by B. anthracis, B. cereus, C. botulinum, C. difficile, C. perfringens, and C. spiroforme consist of components not physically associated in solution that are linked to various diseases in humans, animals, or insects. The “B” components are synthesized as precursors that are subsequently activated by serine-type proteases on the targeted cell surface and/or in solution. Following release of a 20-kDa N-terminal peptide, the activated “B” components form homoheptameric rings that subsequently dock with an “A” component(s) on the cell surface. By following an acidified endosomal route and translocation into the cytosol, “A” molecules disable a cell (and host organism) via disruption of the actin cytoskeleton, increasing intracellular levels of cyclic AMP, or inactivation of signaling pathways linked to mitogen-activated protein kinase kinases. Recently, B. anthracis has gleaned much notoriety as a biowarfare/bioterrorism agent, and of primary interest has been the edema and lethal toxins, their role in anthrax, as well as the development of efficacious vaccines and therapeutics targeting these virulence factors and ultimately B. anthracis. This review comprehensively surveys the literature and discusses the similarities, as well as distinct differences, between each Clostridium and Bacillus binary toxin in terms of their biochemistry, biology, genetics, structure, and applications in science and medicine. The information may foster future studies that aid novel vaccine and drug development, as well as a better understanding of a conserved intoxication process utilized by various gram-positive, spore-forming bacteria.

Binary Bacterial Toxins: Biochemistry, Biology, and Applications of Common Clostridium and Bacillus Proteins

Anthrax lethal factor cleaves the N-terminus of MAPKKs and induces tyrosine/threonine phosphorylation of MAPKs in cultured macrophages.

http://www.jbc.org/content/264/19/11099.full.pdf

Internalization and processing of Bacillus anthracis lethal toxin by toxin-sensitive and -resistant cells.

Anthrax lethal toxin, which consists of two separate proteins, protective antigen (Mr, 82,700) and lethal factor (Mr, approximately 83,000), is cytotoxic to the macrophagelike cell line J774A.1. Removal of calcium from the culture medium protected cells against the action of lethal toxin. Calcium depletion during the binding phase of intoxication afforded only partial protection. Further analysis showed that calcium removal caused some inhibition of protective antigen binding but that it had minimal effect on proteolytic conversion of protective antigen to the active 63-kilodalton fragment and that it had no effect on lethal factor binding. Cells to which lethal toxin had bound in the presence of calcium were protected when transferred to calcium-depleted culture medium, indicating a role for calcium at a postbinding stage. When ammonium chloride is present with lethal toxin, toxin accumulates in intracellular vesicles. Calcium-free medium protected these cells upon removal of the amine block, suggesting that calcium is also required at a step after internalization of lethal toxin. Calcium channel blockers inhibited 45Ca2+ uptake and protected cells against cytotoxicity. Calmodulin inhibitors also protected against the action of lethal toxin, suggesting involvement of calmodulin at a step during intoxication. We conclude that calcium is required at several steps in the intoxication of cells by anthrax lethal toxin.

Calcium is required for the expression of anthrax lethal toxin activity in the macrophagelike cell line J774A.1.

Anthrax lethal toxin, which is composed of two proteins, i.e., protective antigen and lethal factor, is cytolytic to mouse peritoneal macrophages and the macrophage-like cell line J774A.1. After exposure of cells to lethal toxin, inhibition of protein synthesis occurred only slightly before the onset of cytolysis. Thus, cell death did not appear to be due to inhibition of protein synthesis. However, prior treatment of J774A.1 cells with cycloheximide or puromycin, which inhibited protein synthesis, protected them completely against lethal toxin-induced cytolysis, which suggested that continuous protein synthesis is required for the expression of lethal toxin activity. Inhibition of protein synthesis had no appreciable effect on the binding of protective antigen to the cell surface receptor or on proteolytic cleavage of surface-bound protective antigen. Furthermore, inhibition of protein synthesis did not alter the uptake of toxin, which suggested that protein synthesis is required at a later stage of the intoxication process. The protection provided by inhibition of protein synthesis was effective, even up to 1 h after exposure to anthrax lethal toxin. The increased uptake of calcium observed in cells exposed to lethal toxin did not occur when they were protected by blocking protein synthesis. Identifying the protein(s) synthesized during the intoxication process may help to understand the mechanism of cell death produced by anthrax lethal toxin.

https://iai.asm.org/content/iai/62/7/2958.full.pdf

Rakesh Bhatnagar

Papers

Internalization and processing of Bacillus anthracis lethal toxin by toxin-sensitive and -resistant cells.

Calcium is required for the expression of anthrax lethal toxin activity in the macrophagelike cell line J774A.1.

Protein synthesis is required for expression of anthrax lethal toxin cytotoxicity.

Calcium IsRequired fortheExpression ofAnthrax Lethal Toxin Activity intheMacrophagelike CellLineJ774A.1