Diphtheria toxin properties

In the early 1900s, a balanced mixture of diphtheria toxin and antitoxin was found to produce active immunity in both animals and humans. This preparation gained widespread acceptance and protected approximately 85% of recipients. Several years later, diphtheria toxoid was developed by treating the toxin with small amounts of formalin. This process caused the toxin to lose its toxic properties while maintaining its immunogenic properties. In the mid-1920s, the addition of an alum precipitate enhanced the immunogenic properties of the toxoid. 

Classical bacterial exotoxins, such as diphtheria toxin, cholera toxin, clostridial neurotoxins, and the anthrax toxins are enzymes that modify their substrates within the cytosol of mammalian cells. To reach the cytosol, these toxins must first bind to different cell-surface receptors and become subsequently internalized by the cells. To this end, many bacterial exotoxins contain two functionally different domains. The binding (B-) domain binds to a cellular receptor and mediates uptake of the enzymatically active (A-) domain into the cytosol, where the A-domain modifies its specific substrate (see Figure 1). Thus, three important properties characterize the mode of action for any AB-type toxin selectivity, specificity, and potency. Because of their selectivity toward certain cell types and their specificity for cellular substrate molecules, most of the individual exotoxins are associated with a distinct disease. Because of their enzymatic nature, placement of very few A-domain molecules in the cytosol will normally cause a cytopathic effect. Therefore, bacterial AB-type exotoxins which include the potent neurotoxins from Clostridium tetani and C. botulinum are the most toxic substances known today. However, the individual AB-type toxins can greatly vary in terms of subunit composition and enzyme activity (see Table 2). 

The term microbial toxin is usually reserved by microbiologists for toxic substances produced by microorganisms that are of high molecular weight and have antigenic properties toxic compounds produced by bacteria that do not fit these criteria are referred to simply as poisons. Many of the former are proteins or mucoproteins and may have a variety of enzymatic properties. They include some of the most toxic substances known, such as tetanus toxin, botulinus toxin, and diphtheria toxin. Bacterial toxins may be extremely toxic to mammals and may affect a variety of organ systems, including the nervous system and the cardiovascular system. A detailed account of their chemical nature and mode of action is beyond the scope of this volume. 

The formation of oximes and hydrazones can be used to modify proteins due to some attractive properties, that is, some stability in aqueous media and compatibility with many functionalities of proteins [134], Their synthesis can be accomplished through the nucleophilic addition-elimination (condensation) of hydrazines and alkoxyamines with aldehydes and ketones, respectively. Typically, it has been used to modify the N-terminus [135-137] of peptides and proteins. For example, oxime formation has been used to modify BSA and diphtheria toxin with Shigella sonnei O-specific oligosaccharides [138],... 

In the absence of the G protein, PT is able to catalyze the transfer of the ADP-ribose moiety of NAD"" to a water molecule in a reaction known as NAD "-glycohydrolysis (for review, see Locht and Antoine, 1995). This property is also shared with cholera toxin. In contrast, the NAD "-glycohydrolase activities of diphtheria toxin and exotoxin A are much less efficient. The affinity of PT for NAD is in the micromolar range, as evidenced by fluorescent quenching and determina-... 

Sundan A, Olsnes S, Sandvig K, Pihl A (1982) Preparation and properties of chi-maeric toxins prepared from the constituent polypeptides of diphtheria taxin and ricin. Evidence for the entry of ricin A-chain via the diphtheria toxin pathway. J Biol Cbem 257 9733-9739. 

Many antigens currently marketed would be inherently toxic except for the fact they have been stringently detoxified. Some of the most successful and durable vaccines today include toxoided forms of tetanus, pertussis, and diphtheria toxins. During the manufacturing process, these toxoids undergo significant chemical modifications, and their toxic properties are effectively eliminated. The safety is assured with every batch release and defined within safety charac-terization/quality control tests, which are not discussed in this chapter. 

The other elongation factors, EF-ipy and EF-2, are involved, respectively, in the posthydrolytic exchange of GDP with GTP and in the translocation of peptidyl-tRNA on the ribosome. Of these, EF-2 (95 kDa) has a unique characteristic in the form of a histidine residue at position 715 modified into diphthamide, as a result of which it can be ADP-ribosylated either endogenously or by bacterial toxins such as diphtheria toxin (Riis et al., 1990b). EF-3, which is present in some yeast and fungal species, is a 125 kDa polypetide chain. A unique property of EF-3 is its ability to function with any of the three purine nucleosides (A, G, and I), perhaps to facilitate the interaction of EF-1 a, GTP, and aminoacyl-tRNA. The requirement for EF-3, even in vitro, is an exclusive property of yeast ribosomes. When yeast factors are tested with mammalian ribosomes, EF-3 has no effect (Riis et al., 1990). 

Uchida, T Pappenheimer, A. M. J., and Greany, R. (1973) Diphtheria toxin and related proteins I. Isolation and properties of mutant proteins serologically related to diphtheria toxin. J. Biol. Chem. 248(11), 3838-3844. 

Many of the differences between translation in prokaryotes and eukaryotes can be seen in the response to inhibitors of protein synthesis and to toxins. The antibiotic chloramphenicol (a trade name is Ghloromycetin) binds to the A site and inhibits peptidyl transferase activity in prokaryotes, but not in eukaryotes. This property has made chloramphenicol useful in treating bacterial infections. In eukaryotes, diphtheria toxin is a protein that interferes with protein synthesis by decreasing the activity of the eukaryotic elongation factor eEF2. 

Van Ness BG, Howard JB, Bodley JW (1980) ADP-ribosylation of elongation factor 2 by diphtheria toxin. Isolation and properties of the novel ribosyl-amino acid and its hydrolysis products. J Biol Chem 255 10717-10720... 

Iglewski WJ, Lee H (1983) Purification and properties of an altered form of elongation factor 2 from mutant cells resistant to intoxication by diphtheria toxin. Eur J Biochem 134 237-240... 

The inhibition of protein synthesis in Chinese hamster cells by diphtheria toxin is antagonized by concanavalin A, succinylated concanavalin A, and wheat germ agglutinin. The effects are reversed by methyl a-D-manno-pyranoside in the first two cases and by 2-acetamido-2-deoxy-D-glucose in the last. Thus diphtheria toxin appears to have the saccharide binding properties of a lectin. 

Some of the physical and chemical properties of tetanus, botulinus, and diphtheria toxins, together with comparable data for crystalline human serum albumin, are summarized in Table II. 

Property Diphtheria toxin Tetanus toxin Botulinus toxin, type A Human serum albumin ... 

Toxoids (or antitoxins)—A toxoid is a famed toxin which is treated chemically so that it loses the poisonous or toxic properties but still retains the power to stimulate the body cells to form the appropriate antibody. Typical toxoids are diphtheria toxoid and tetanus toxoid. 

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