Clostridial Specificity

Important members of this toxin family are Clostridium difficile toxins A and B, which are implicated in antibiotics-associated diarrhea and pseudomembranous colitis. The large clostridial cytotoxins are single-chain toxins with molecular masses of 250-308 kDa. The enzyme domain is located at the N terminus. The toxins are taken up from an acidic endosomal compartment. They glucosylate RhoA at Thr37 also, Rac and Cdc42 are substrates. Other members of this toxin family such as Clostridium sordellii lethal toxin possess a different substrate specificity and modify Rac but not Rho. In addition, Ras subfamily proteins (e.g., Ras, Ral, and Rap) are modified. As for C3, they are widely used as tools to study Rho functions [2] [4]. 

The light chains of the clostridial neurotoxins are metalloproteases with exclusive specificity for neuronal SNAREs. TeNT, BoNTs B,D,F, and G cleave synapto-brevin, BoNTs A and E SNAP-25, and BoNT/Cl syntaxin, and to a lesser extent also SNAP-25. Cleavage of any of the SNAREs causes complete and irreversible block of synaptic transmission. 

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). 

In spite of these various differences, there is one respect in which C2 toxin is akin to other clostridial toxins, such as botulinum neurotoxin and tetanus toxin its ability to act at low concentrations to produce a specific and irreversible effect on many types of cells can be translated into a benefit. This toxin has great potential as a research toal to study eukaryotic cell biology. 

The extreme toxicity of clostridial neurotoxins (CNTs) derives from their absolute neurospecificity as well as from catalytic activity. TeTx and BoNTs bind specifically to the neuromuscular junction (NMJ) of motor neurons. The identity of the receptor(s) on the presynaptic membrane is unknown, but their extreme toxicity suggests that the binding affinity to the cognate receptor must be very high. The receptor-bound toxin is internalized at the presynaptic membrane of the NMJ and gains access to the neuronal cytosol. Here it blocks the release of acetylcholine (ACh), causing a flaccid paralysis (Simpson,... 

Fig. 3. Specificity and sites of cleavage of the clostridial neurotoxins. VAMP is bound to the SSV membrane through a single transmembrane domain (black box), with the majority of the protein exposed to the cytoplasm. In addition, VAMP contains an amino-terminal domain rich in proline (hatched box). SNAP-25 and syn-taxin are bound to the target membrane via palmitoylation (SNAP-25) or via a single transmembrane domain (syntaxin). TeTx and BoNT/B, D, F or G act on the conserved central portion of VAMP and release its amino-terminal part into the cytosol. The sequences indicate the peptide bonds cleaved by CNTs on rat VAMP-1 and VAMP-2. BoNT/A and E cleave SNAP-25 at the carboxyl terminus, with the release of nine and twenty-six residues peptides respectively. BoNT/C also cleaves SNAP-25 at the carboxy-terminus, and cleaves syntaxin at a single site near the cytosolic membrane surface. The action of TeTx and BoNT/B, C, D, F and G causes the release of a large portion of the cytosolic domain of VAMP and syntaxin. Conversely, only a small segment of SNAP-25 is released by the selective proteolysis of BoNT/A, C and E...

The clostridial neurotoxins responsible for tetanus and botulism form a new group of zinc-endopeptidases endowed with peculiar properties. They are produced as inactive precursors which are activated by specific proteolysis, followed by intracellular reduction of a single di-... 

Figure 2 summarizes the procedure to monitor the blockade of neurotransmitter release and the specific proteolytic activity of clostridial toxins on brain synaptosomes. 

Fig. 2. Schematic drawing that summarizes the procedure for monitoring the blockade of neurotransmitter release from brain synaptosomes, followed by immu-noblot analysis to detect specific proteolytic activity of clostridial neurotoxins on poisoned synaptosomes...

Note that pH 11 was used for the purely chemical titration in Fig. 8 (A) in order to reach a sufficiently negative potential while in the electrochemical titration in fig. 8 (B) pH 9.5 was sufficient to extend the potential to -700 mV. Previously, Lozier and Butler had performed a redox titration ofthe PS-I reaction and obtained results very similar to those shown in Fig. 8 (A). These workers, using the Clostridial H/HV hydrogenase system as the reductant and l,l -trimethylene-2-2 -dipyridylium dibromide as the mediator, achieved specific potentials by gradually varying the pH between 8 and 10 to effect reduction ofthe photosystem-I electron acceptors. 

Fe Protein. Characteristics of the Fe protein from Azotobacter 101) and Clostridia (94, 108) are summarized in Table VII. Clostridial Fe protein is stated to be 90-95% pure and the absence of tryptophan (102) suggests that almost all of the contaminating proteins have been removed. Recent preparations of Azotobacter Fe protein show specific activities even higher than those of clostridial Fe protein (101). The clostridial protein has a molecular weight of 39,000 and contains 2-3 Fe and 2 S atoms (94, 108). Molybdenum is absent, no ESR is detectable in the native protein (102) and, surprisingly, a resonance at g value of 1.94 has not been observed on reduction. All other purified iron-sulfur proteins with the exception of the atypical HIPIP and rubredoxin exhibit resonance in this area on reduction. One may suggest that the Fe protein is an atypical iron-sulfur protein or requires additional examination at 4°K for a resonance at g = 1.94. The individual protein has no activity alone but has all N2ase activities in combination with the Mo-Fe protein. 

Clostridial ferredoxin was discovered by virtue of its requirement in the oxidation of pyruvate by the clostridial species specifically involved in a nitrogen-fixing system (44). We can now interpret the requirement for pyruvate oxidation as a reaction leading to the formation of reduced ferredoxin, which in turn acted in the reduction of the various substrates, including N2 (45). But there are many other substrates which can be reduced by the reduced ferredoxin—i.e., in the presence of the appropriate enzyme. For example, nitrite may be reduced to ammonia. Reduced ferredoxin can be formed by nonphotosynthetic bacteria by reactions other than puruvate oxidation it can also be formed by the hydrogenase, or by the oxidation of a purine. 

Demonstration of this activity was initially difficult because the toxins are very specific for their substrates. However, it is now clear that (a) the clostridial neurotoxins express proteolytic activity and (b) this activity is absolutely required for toxicity.30 The substrate proteins for this action appear to be part of a hetero-oligomeric assembly associated with the synaptic vesicles. Interestingly, the specific target site for cleavage seems to be different for each serotype of the botulinum toxins in some cases, different locations on the same protein in others, different proteins of the assembly.30 The basis of this marked specificity is not yet clear and remains fascinating to scientists interested in neurosecretion. 

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