Botulinum neurotoxin serotype C (BoNT/C) is a 150-kDa protein produced by Clostridium botulinum, which causes animal botulism. In contrast to the other botulinum neurotoxins that contain one atom of zinc, highly purified preparations of BoNT/C bind two atoms of zinc per toxin molecule. BoNT/C is a zinc-endopeptidase that cleaves syntaxin 1A at the Lys253-Ala254 and syntaxin 1B at the Lys252-Ala253 peptide bonds, only when they are inserted into a lipid bilayer. The other Lys-Ala bond present within the carboxyl-terminal region is not hydrolyzed. Syntaxin isoforms 2 and 3 are also cleaved by BoNT/C, while syntaxin 4 is resistant. These data suggest that BoNT/C recognizes a specific spatial organization of syntaxin, adopted upon membrane insertion, which brings a selected Lys-Ala peptide bond of its carboxyl-terminal region to the active site of this novel metalloproteinase.

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Clostridium botulinum type B neurotoxin has been shown to be a zinc endopeptidase specific for vesicle-associated membrane protein (VAMP). A synthetic peptide of h u m d r a t VAMP-2 [ VAMP-2-(60-94)] is cleaved by the neurotoxin with the same specificity as that demonstrated for the membrane-associated protein (at the Gln76-Phe77 bond) and has been used to study the properties of the endopeptidase activity of the neurotoxin. Cleavage of the VAMP-2 peptide was demonstrated by both botulinum type B neurotoxin (K, = 3.3X10-4M) and by its purified light subunit (K, = 3.5X10-4M). The endopeptidase displayed a pH optimum of 7.0-7.5 and was inhibited by greater than 0.2 M NaCl and greater than 0.05 M sodium phosphate. Neurotoxin which had been inactivated by dialysis against EDTA could be re-activated by incubation with various divalent cations, notably Znz+ and Cu2+. The substrate specificity of botulinum type B neurotoxin was studied using various analogues of VAMP-2 (60-94). The neurotoxin cleaved selectively to the N-terminal side of phenylalanine and tyrosine; no activity was observed with either leucine, valine or alanine in the P: position. The properties of the P, amino acid were less critical; the neurotoxin cleaving the Cterminus of glutamine, asparagine and alanine. A substrate analogue with valine in the P, position corresponding to the sequence of rat VAMP-1 was not cleaved. The rate of cleavage of a substrate analogue representing the sequence of human VAMP-1, however, was more than twofold that of the VAMP-2 peptide. The properties and substrate specificity of botulinum type B neurotoxin suggest that the toxin represents a novel class of endopeptidase which requires a specific peptide substrate conformation for the expression of proteolytic activity.

Clostridium difficile is a major and growing problem as a hospital-associated infection that can cause severe, recurrent diarrhea. The mechanism by which the bacterium colonizes the gut during infection is poorly understood but undoubtedly involves protein components within the surface layer (S-layer), which play a role in adhesion. In C. difficile, the S-layer is composed of two principal components, the high and low molecular weight S-layer proteins, which are formed from the post-translational cleavage of a single precursor, SlpA. In the present study, we demonstrate that a recently characterized cysteine protease, Cwp84 plays a role in maturation of SlpA. Using a gene knockout approach, we show that inactivation of the Cwp84 gene in C. difficile 630⌬Erm results in a bacterial phenotype in which only immature, single chain SlpA comprises the S-layer. The Cwp84 knock-out mutants (CD⌬Cwp84) displayed significantly different colony morphology compared with the wild-type strain and grew more slowly in liquid medium. SlpA extracted from CD⌬Cwp84 was readily cleaved into its mature subunits by trypsin treatment. Addition of trypsin to the growth medium also cleaved SlpA on CD⌬Cwp84 and increased the growth rate of the bacterium in a dose-dependent manner. Using the hamster model for C. difficile infection, CD⌬Cwp84 was found to be competent at causing disease with a similar pathology to the wild-type strain. The data show that whereas Cwp84 plays a role in the cleavage of SlpA, it is not an essential virulence factor and that bacteria expressing immature SlpA are able to cause disease.

Recent data suggest that botulinum type-B neurotoxin is a protease which acts on vesicleassociated membrane protein, isoform 2 (VAMP-2). In this report, botulinum type-B neurotoxin is shown to cleave a synthetic fragment (HV62) of VAMP-2, corresponding to the bulk of the hydrophillic domain (amino acids 33-94). The neurotoxin acts at a single site between Gln76 and Phe77. Little or no proteolytic activity by botulinum type-B neurotoxin was observed with peptides containing 7, 10 or 20 amino acids spanning the site of cleavage. The proteolytic action of neurotoxin was strongly inhibited by EDTA and o-phenanthroline whereas captopril and phosphoramidon were ineffective. A series of model peptide substrates were synthesised in order to define the smallest VAMP-2 fragment to be cleaved by botulinum type-B neurotoxin. Data obtained from these substrates suggest that the neurotoxin belongs to a novel class of zinc-endoprotease; more than 12 amino acid residues are required on both the NH,-and COOH-terminal side of the cleavage site for optimal proteolytic activity. The results demonstrate that no other components of cellular vesicles are required for the specific action of the neurotoxin on VAMP-2. The data further show that the highly specific action of the neurotoxin is not dictated solely by the properties of the amino acid residues at the cleavage site but is also dependent on amino acid sequences distal to its site of action.

Bacteria possess complex and varying cell walls with many surface exposed proteins. Sortases are responsible for the covalent attachment of specific proteins to the peptidoglycan of the cell wall of Gram‐positive bacteria. Sortase A of Staphylococcus aureus, which is seen as the archetypal sortase, has been shown to be essential for pathogenesis and has therefore received much attention as a potential target for novel therapeutics. Being widely present in Gram‐positive bacteria, it is likely that other Gram‐positive pathogens also require sortases for their pathogenesis. Sortases have also been shown to be of significant use in a range of industrial applications. We review current knowledge of the sortase family in terms of their structures, functions and mechanisms and summarize work towards their use as antibacterial targets and microbiological tools.

Botulinum neurotoxins consist of a metalloprotease linked via a conserved interchain disulfide bond to a heavy chain responsible for neurospecific binding and translocation of the enzymatic domain in the nerve terminal cytosol. The metalloprotease activity is enabled upon disulfide reduction and causes neuroparalysis by cleaving the SNARE proteins. Here, we show that the thioredoxin reductase-thioredoxin protein disulfide-reducing system is present on synaptic vesicles and that it is functional and responsible for the reduction of the interchain disulfide of botulinum neurotoxin serotypes A, C, and E. Specific inhibitors of thioredoxin reductase or thioredoxin prevent intoxication of cultured neurons in a dose-dependent manner and are also very effective inhibitors of the paralysis of the neuromuscular junction. We found that this group of inhibitors of botulinum neurotoxins is very effective in vivo. Most of them are nontoxic and are good candidates as preventive and therapeutic drugs for human botulism.

Tetanus and botulinum neurotoxins type B and G are zinc-endopeptidases of remarkable specificity. They recognize and cleave a synaptic vesicle-associated membrane protein (VAMP)/synaptobrevin, an essential protein component of the vesicle docking and fusion apparatus. VAMP contains two copies of a nine-residue motif, also present in SNAP-25 (synaptosomal-associated protein of 25 kDa) and syntaxin, the two other substrates of clostridial neurotoxins. This motif was suggested to be a determinant of the target specificity of neurotoxins. Antibodies raised against this motif cross-react among VAMP, SNAP-25, and syntaxin and inhibit the proteolytic activity of the neurotoxins. Moreover, the various neurotoxins cross-inhibit each other's proteolytic action. The role of the three negatively charged residues of the motif in neurotoxin recognition was probed by sitedirected mutagenesis. Substitution of acidic residues in both copies of the VAMP motif indicate that the first one is involved in tetanus neurotoxin recognition, whereas the second one is implicated in binding botulinum B and G neurotoxins. These results suggest that the two copies of the motif have a tandem association in the VAMP molecule.Tetanus neurotoxin (TeNT) 1 and botulinum neurotoxins (BoNTs, seven types from A to G) are three-domain protein toxins that bind selectively to the neuronal presynaptic membrane. They are internalized inside intracellular compartments from which the amino-terminal 50-kDa domain (termed L chain) enters into the cytosol (1-4). The L chains of TeNT and BoNTs are zinc-endopeptidases that cleave specifically three proteins of the neuroexocytosis apparatus, thereby blocking neurotransmitter release (4 -7). TeNT and BoNT/B, BoNT/D, BoNT/F, and BoNT/G recognize and cleave specifically a synaptic vesicle-associated membrane protein (VAMP, also referred to as synaptobrevin) at different single peptide bonds (4, 8 -12). BoNT/A and BoNT/E specifically recognize and cut SNAP-25 (synaptosomal-associated protein of 25 kDa) at two different peptide bonds near the COOH terminus (10, 13, 14), whereas BoNT/C cleaves syntaxin (15, 16) and . VAMP, SNAP-25, and syntaxin are collectively termed SNARE proteins, because they act as receptors of soluble Nethylmaleimide-sensitive factor accessory proteins, involved in vesicle-membrane fusion (5-7).Sequence comparison of the L chains of the eight clostridial neurotoxins show strong similarities (20), which are even more extensive at the level of predicted secondary structure (21). These similarities suggest that they derive from a common ancestral metalloproteinase. On this basis, to account for their different substrate specificity, we considered the possibility that the three SNAREs contain a common neurotoxin recognition site in addition to the cleavage sites specific for each neurotoxin type. We identified a nine-residue-long motif (SNARE motif) present in eukaryotes only in the three proteins known to be proteolytic substrates of the neurotoxins (22). The SNARE motif is included within regions...