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.

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.

ADP-ribosylation is one of the favored modes of cell intoxication employed by several bacteria. Clostridium difficile is recognized to be an important nosocomial pathogen associated with considerable morbidity and attributable mortality. Along with its two well known toxins, Toxin A and Toxin B, it produces an ADP-ribosylating toxin that targets monomeric actin of the target cell. Like other Clostridial actin ADP-ribosylating toxins, this binary toxin, known as C. difficile toxin (CDT), is composed of two subunits, CDTa and CDTb. In this study, we present high resolution crystal structures of CDTa in its native form (at pH 4.0, 8.5, and 9.0) and in complex with ADP-ribose donors, NAD and NADPH (at pH 9.0). The crystal structures of the native protein show "pronounced conformational flexibility" confined to the active site region of the protein and "enhanced" disorder at low pH, whereas the complex structures highlight significant differences in "ligand specificity" compared with the enzymatic subunit of a close homologue, Clostridium perfringens iota toxin. Specifically in CDTa, two of the suggested catalytically important residues (Glu-385 and Glu-387) seem to play no role or a less important role in ligand binding. These structural data provide the first detailed information on protein-donor substrate complex stabilization in CDTa, which may have implications in understanding CDT recognition.Clostridium difficile infection is a major problem as a healthcare-associated infection. The bacterium causes nosocomial, antibiotic-associated diarrhea and pseudomembranous colitis in patients treated with broad spectrum antibiotics (1-3). Elderly patients are most at risk from these potentially life-threatening diseases, and incidents of hospital infection have increased dramatically over the last 10 years.Strains of C. difficile produce a variety of virulence factors, notable among which are several protein toxins: Toxin A, Toxin B (4 -6), and, in some strains, the binary toxin CDT, 3 which is similar to Clostridium perfringens iota toxin and Clostridium botulinum C2 toxin (7-9). Toxins A and B are large protein cytotoxins that play a key role in the pathology of infection and most probably are involved in the gut colonization process. Outbreaks of C. difficile infection have been reported with Toxin A-negative/Toxin B-positive strains, and a recent report (10) suggests that Toxin B plays a major role in the disease pathology. Little is presently known about the contribution of the binary toxin to C. difficile infection.CDT binary toxin belongs to the family of actin-specific ADP-ribosylating toxin (ADPRT) (for a recent review see Ref.11), composed of two independently produced components: a transport component of 99 kDa (CDTb) that facilitates translocation of the enzymatic component of 49 kDa (CDTa) into the target cell that is capable of transferring ADP-ribose group of NAD/NADPH to monomeric actin molecules in target cells (9,12,13). This irreversible modification of G-actin at 14) blocks its polymerization and thus...

Clostridium difficile, a highly infectious bacterium, is the leading cause of antibiotic-associated pseudomembranous colitis. In 2009, the number of death certificates mentioning C. difficile infection in the U.K. was estimated at 3933 with 44% of certificates recording infection as the underlying cause of death. A number of virulence factors facilitate its pathogenicity, among which are two potent exotoxins; Toxins A and B. Both are large monoglucosyltransferases that catalyse the glucosylation, and hence inactivation, of Rho-GTPases (small regulatory proteins of the eukaryote actin cell cytoskeleton), leading to disorganization of the cytoskeleton and cell death. The roles of Toxins A and B in the context of C. difficile infection is unknown. In addition to these exotoxins, some strains of C. difficile produce an unrelated ADP-ribosylating binary toxin. This toxin consists of two independently produced components: an enzymatic component (CDTa) and the other, the transport component (CDTb) which facilitates translocation of CDTa into target cells. CDTa irreversibly ADP-ribosylates G-actin in target cells, which disrupts the F-actin:G-actin equilibrium leading to cell rounding and cell death. In the present review we provide a summary of the current structural understanding of these toxins and discuss how it may be used to identify potential targets for specific drug design.

Treatment of Clostridium difficile is a major problem as a hospital-associated infection which can cause severe, recurrent diarrhea. The currently available antibiotics are not effective in all cases and alternative treatments are required. In the present study, an ovine antibody-based platform for passive immunotherapy of C. difficile infection is described. Antibodies with high toxin-neutralizing titers were generated against C. difficile toxins A and B and were shown to neutralize three sequence variants of these toxins (toxinotypes) which are prevalent in human C. difficile infection. Passive immunization of hamsters with a mixture of toxin A and B antibodies protected them from a challenge with C. difficile spores in a dose-dependent manner. Antibodies to both toxins A and B were required for protection. The administration of toxin A and B antibodies up to 24 h postchallenge was found to reduce significantly the onset of C. difficile infection compared to nonimmunized controls. Protection from infection was also demonstrated with key disease isolates (ribotypes 027 and 078), which are members of the hypervirulent C. difficile clade. The ribotype 027 and 078 strains also have the capacity to produce an active binary toxin and these data suggest that neutralization of this toxin is unnecessary for the management of infection induced by these strains. In summary, the data suggest that ovine toxin A and B antibodies may be effective in the treatment of C. difficile infection; their potential use for the management of severe, fulminant cases is discussed.

The enzyme porphobilinogen deaminase (PBGD; hydroxymethylbilane synthase; EC 2.5.1.61) catalyses a key early step of the haem- and chlorophyll-biosynthesis pathways in which four molecules of the monopyrrole porphobilinogen are condensed to form a linear tetrapyrrole. The active site possesses an unusual dipyrromethane cofactor which is extended during the reaction by the sequential addition of the four substrate molecules. The cofactor is linked covalently to the enzyme through a thioether bridge to the invariant Cys254. Until recently, structural data have only been available for the Escherichia coli and human forms of the enzyme. The expression of a codon-optimized gene for PBGD from Arabidopsis thaliana (thale cress) has permitted for the first time the X-ray analysis of the enzyme from a higher plant species at 1.45 Å resolution. The A. thaliana structure differs appreciably from the E. coli and human forms of the enzyme in that the active site is shielded by an extensive well defined loop region (residues 60-70) formed by highly conserved residues. This loop is completely disordered and uncharacterized in the E. coli and human PBGD structures. The new structure establishes that the dipyrromethane cofactor of the enzyme has become oxidized to the dipyrromethenone form, with both pyrrole groups approximately coplanar. Modelling of an intermediate of the elongation process into the active site suggests that the interactions observed between the two pyrrole rings of the cofactor and the active-site residues are highly specific and are most likely to represent the catalytically relevant binding mode. During the elongation cycle, it is thought that domain movements cause the bound cofactor and polypyrrole intermediates to move past the catalytic machinery in a stepwise manner, thus permitting the binding of additional substrate moieties and completion of the tetrapyrrole product. Such a model would allow the condensation reactions to be driven by the extensive interactions that are observed between the enzyme and the dipyrromethane cofactor, coupled with acid-base catalysis provided by the invariant aspartate residue Asp95.

Sortase enzymes are responsible for covalent anchoring of specific proteins to the peptidoglycan of the cell wall of gram-positive bacteria. In some gram-positive bacteria (e.g. Staphylococcus aureus), sortases have been found to be essential for pathogenesis and their inhibitors are under development as potential novel therapeutics. Here we provide the first report on the structural characterisation of the C. difficile sortase. An active site mutant was crystallised and its structure determined to 2.55 Å by X-ray diffraction to provide structural insight into its catalytic mechanism. In order to elucidate the role of the sortase in the cell wall biogenesis, a C. difficile sortase knockout strain was constructed by intron mutagenesis. Characterisation of this mutant led to the discovery that the putative adhesin CD0386 is anchored to the peptidoglycan of C. difficile by the sortase SrtB and that an SPKTG peptide motif is involved in the transpeptidation reaction with the C. difficile peptidoglycan. In an animal model for C. difficile infection, the SrtB mutant caused disease at a similar rate of onset as the wild type strain. In conclusion, our detailed study shows that the SrtB enzyme from C. difficile does not play an essential role in pathogenesis.