Caister Academic Press

Clostridial toxins

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The genus Clostridium represents a heterogeneous group of anaerobic spore-forming bacteria, comprising prominent toxin-producing species, such as C. difficile, C. botulinum, C. tetani and C. perfringens. C. tetani and C. botulinum produce the most potent biological toxins known to affect humans.

Botulinum and Tetanus Neurotoxins
Botulinum neurotoxins (BoNT) and tetanus toxin (TeNT) are potent toxins which are responsible for severe diseases, botulism and tetanus, in men and animals. BoNTs induce a flaccid paralysis, whereas TeNT causes a spastic paralysis. Both toxins are zinc-dependent metalloproteases, which specifically cleave one of the three proteins (VAMP, SNAP25, and syntaxin) forming the SNARE complex within target neuronal cells which have a critical function in the release of neurotransmitter. BoNTs inhibit the release of acetylcholine at peripheral cholinergic nerve terminals, whereas TeNT blocks neurotransmitter release at central inhibitory interneurons. Only a single form of TeNT is known, but BoNTs are divided in 7 toxinotypes and various subtypes, which differ in amino acid sequences and immunological properties. In contrast to TeNT, BoNTs are associated to non-toxic proteins (ANTPs) to form highly stable botulinum complexes. TeNT is produced by Clostridium tetani, and BoNTs by Clostridium botulinum and atypical strains of Clostridium barati and Clostridium butyricum. The genes encoding the neurotoxin and ANTPs are clustered in a DNA segment, called botulinum locus, which is located on chromosome, plasmid or phage. Neurotoxin synthesis is a highly regulated process, which occurs in late exponential growth phase and beginning of stationary phase, and which is dependent of alternative sigma factors (BotR or TetR). BotR and TetR are related to other clostridial sigma factors, TcdR and UviA, which are involved in the control of Clostridium difficile toxins A and B, and Clostridium perfringens bacteriocin, respectively. BotR, TetR, TcdR and UviA form a new subgroup of RNA polymerase sigma factors.

Clostridium perfringens Enterotoxin
Clostridium perfringens enterotoxin (CPE) causes the intestinal symptoms of a common food-borne illness and ~5-15% of all antibiotic-associated diarrhea cases. In food poisoning isolates, the enterotoxin gene (cpe) is usually present on the chromosome, while cpe is carried by conjugative plasmids in antibiotic-associated diarrhea isolates. CPE action involves its binding to claudin receptors, oligomerization/prepore formation, and prepore insertion to form a functional pore that kills cells by apoptosis or oncosis. The C-terminal half of CPE mediates receptor binding, while its N-terminal half is required for oligomerization. CPE/CPE derivatives are being explored for cancer therapy/diagnosis and improved drug delivery.

The Cholesterol-dependent Cytolysins and Clostridium septicum α-Toxin
Two classes of pore-forming toxins of the clostridia are represented by the cholesterol-dependent cytolysins (CDCs) and the Clostridium septicum α-toxin. The CDCs are found in a wide variety of clostridial species, but are also found in many species from other Gram-positive genera. As a result, various CDCs have evolved specific traits that appear to enhance their ability to complement the pathogenic mechanism of a specific bacterial species. In contrast, closely related toxins to C. septicum α-toxin (AT) have not been found in other species of the clostridia, although C. perfringens epsilon toxin appears to be distantly related. Remarkably, distant relatives of AT have been found in species of Gram-negative bacteria as well as certain species of mushrooms and the enterolobin tree seed. Although the CDCs appear to be restricted to Gram-positive bacterial pathogens it has recently been shown that the unusual protein fold of their membrane-penetrating domain is present in proteins of the eukaryotic complement membrane attack complex. Both toxins penetrate the membrane by the use of a β-barrel pore but differ significantly in their pore-forming mechanisms. The contribution of both classes of toxins to disease is not yet well understood for the clostridia. It is clear that they play important, but likely different roles in clostridial disease.

Binary Bacterial Toxins
Several proteins from Gram-positive, spore-forming bacilli use a synergistic binary mechanism for intoxicating eukaryotic cells. These toxins include Clostridium botulinum C2 toxin, Clostridium difficile toxin (CDT), Clostridium perfringens iota (ι) toxin, and Clostridium spiroforme toxin (CST). Furthermore, closely related Bacillus species such as Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis produce strikingly similar binary toxins. As per existing literature, these latter proteins have provided a "model" for the clostridial binary toxins. Each of these clostridial and bacillus binary toxins consists of distinct enzymatic "A" and binding "B" proteins that work in concert. Conservation of a basic intoxication theme between different genera clearly suggests retention of an evolutionarily successful mechanism promoting bacterial survival and dissemination throughout Nature.

Group I and II Clostridium botulinum
Clostridium botulinum, producing highly potent botulinum neurotoxin, is a diverse species consisting of four genetically and physiologically distinct groups (Groups I-IV) of organisms. Groups I and II C. botulinum produce A, B, E, and/or F toxins which cause human botulism. In addition, some strains of Clostridium butyricum and Clostridium barati produce type E and F toxins, respectively, and have thus been related to human illness. Human botulism appears in five different forms, such as the classical food botulism, infant botulism, wound botulism, adult infectious botulism, and iatrogenic botulism. Typical of all forms of human botulism is descending flaccid paralysis which may lead to death upon respiratory muscle failure. While the research and diagnostics of botulinum neurotoxigenic clostridia and botulism were based on toxin detection by the mouse bioassay until mid 1990's, the subsequent development of molecular detection and typing assays enabled rapid, sensitive, specific, and ethically acceptable molecular epidemiological detection, identification and strain characterization of these organisms, increasing our understanding of the epidemiology of botulinum neurotoxigenic clostridia and botulism.

C. difficile large clostridial toxins
Clostridium difficile, as all clostridia, is a toxin producing microorganism and the toxins are the main virulence factors. In the early eighties it was clear that two large toxins are produced by the bacterium and epidemiological studies have indicated that strains either produce both toxins (toxin A, TcdA, and toxin B, TcdB) or none of them. Toxigenic strains were usualy associated with the disease, while nontoxigenic were not. This simple situation changed as strains producing only TcdB or strains producing an additional toxin (binary toxin CDT) were described. Such strains with unusual toxin production pattern were subsequently found to have changes in the genomic PaLoc region encoding the toxins TcdA and TcdB. These changes are the basis for a method that distinguish C. difficile strains into toxinotypes. The variability of genes coding for large clostridial toxins (LCTs) has consequences in laboratory diagnosis, changes in understanding of the role of both toxins in pathogenesis, in structure function relationships and in the understanding of the evolutions of LCTs.

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