Bacterial toxins: a table of lethal amounts.

ADP-ribosylation of regulatory proteins is an important pathological mechanism by which various bacterial toxins affect eukary-otic cell functions. While diphtheria toxin catalyses the ADP-ribosylation of elongation factor 2, which results in inhibition of protein synthesis, cholera toxin and pertussis toxin ADP-ribosylate Ns and Ni respectively, the GTP-binding regulatory components of the adenylate cyclase system, thereby modulating the bidirectional hormonal regulation of the adenylate cyclase1,2Botulinum C2 toxin is another toxin which has been reported to possess ADP-ribosyltransferase activity3. This extremely toxic agent is produced by certain strains of Clostridium botulinum4 and induces hypotension5, an increase in intestinal secretion6 vascular permeability7 and haemorrhaging in the lungs5. In contrast to botulinum neurotoxins, the botulinum C2 toxin apparently lacks any neurotoxic effects5. Here we report that botulinum C2 toxin ADP-ribosylates a protein of relative molecular mass 43,000 (43K) in intact cells and in cell-free preparations. We present evidence that the 43K protein substrate is actin, which is apparently mono-AD P-ribosylated by the toxin. Botulinum C2 toxin also ADP-ribosylated purified liver G-actin, whereas liver F-actin was only poorly ADP-ribosylated and skeletal muscle actin was not ADP-ribosylated in either its G form or its F form. ADP-ribosylation of liver G-actin by botulinum C2 toxin resulted in a drastic reduction in viscosity of actin polymerized in vitro.

Botulinum C2 toxin ADP-ribosylates actin

https://mmbr.asm.org/content/mmbr/60/1/167.full.pdf

Enteric bacterial toxins: mechanisms of action and linkage to intestinal secretion.

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

By screening possible ADP-ribosyltransferase activities in culture supernatants from various Clostridium species, we have found one Clostridium difficile strain (CD196) (isolated in our laboratory) that is able to produce, in addition to toxins A and B, a new ADP-ribosyltransferase that was shown to covalently modify cell actin as Clostridium botulinum C2 or Clostridium perfringens E iota toxins do. The molecular weight of the CD196 ADP-ribosyltransferase (CDT) was determined to be 43 kilodaltons, and its isoelectric point was 7.8. No cytotoxic activity on Vero cells or lethal activity upon injection in mice was associated with this enzyme. CDT was neither related to C. difficile A or B toxins nor to C. botulinum C2 toxin component I. However, Vero cells cultivated in the presence of C. difficile B toxin had a lower amount of actin able to be ADP-ribosylated by CDT or C2 toxin in vitro. Antibodies raised against CDT reacted by immunoblot analysis with a 43-kilodalton protein of C. perfringens type E culture supernatant producing the iota toxin. Images

Actin-specific ADP-ribosyltransferase produced by a Clostridium difficile strain.

Two dissimilar proteins, designated as components I and II, of botulinum C2 toxin elaborated by strain 92-13 were purified to a homogeneous state. The molecular weights determined by sodium dodecyl sulfate gel electrophoresis were 55,000 for component I and 105,000 for component II. Whereas each component showed no or feeble toxicity even after being treated with trypsin, the toxicity was elicited when these two components were mixed and trypsinized. The toxicity of the mixture of components I and II at a ratio of 1:2.5 on a protein basis was 2.2 X 10(4) mouse intraperitoneal 50% lethal doses per mg of protein and increased by 2,000 times or more when treated with trypsin. These results indicate that the molecular characteristics of botulinum C2 toxin differ from those of the toxin of Clostridium botulinum types A through F in that C2 toxin is constructed with two separate protein components, which are not covalently held together, and that its toxicity is elicited by cooperation of the two components.

/pdf/purification-and-characterization-of-two-components-of-383jc03fiq.pdf

G Sakaguchi

Papers

Purification and characterization of two components of botulinum C2 toxin.