April K. Roberts

Cwp84, a Surface-associated Cysteine Protease, Plays a Role in the Maturation of the Surface Layer of Clostridium difficile

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 knock-out approach, we show that inactivation of the Cwp84 gene in C. difficile 630ΔErm results in a bacterial phenotype in which only immature, single chain SlpA comprises the S-layer. The Cwp84 knock-out mutants (CDΔCwp84) displayed significantly different colony morphology compared with the wild-type strain and grew more slowly in liquid medium. SlpA extracted from CDΔCwp84 was readily cleaved into its mature subunits by trypsin treatment. Addition of trypsin to the growth medium also cleaved SlpA on CDΔCwp84 and increased the growth rate of the bacterium in a dose-dependent manner. Using the hamster model for C. difficile infection, CDΔCwp84 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.

Super toxins from a super bug: structure and function of Clostridium difficile toxins

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.

Structural Basis for Substrate Recognition in the Enzymatic Component of ADP-ribosyltransferase Toxin CDTa from Clostridium difficile

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.

Expression, purification and cell cytotoxicity of actin-modifying binary toxin from Clostridium difficile

Clostridium difficile infection (CDI) is a serious problem within the healthcare environment where the bacterium causes symptoms ranging from mild diarrhoea to life-threatening colitis. In addition to its principal virulence factors, Toxin A and Toxin B, some C. difficile strains produce a binary toxin (CDT) composed of two sub-units namely CDTa and CDTb that are produced and secreted from the cell as two separate polypeptides. Once in the gut these fragments have the potential to combine to form a potent cytotoxin whose role in the pathogenesis of CDI is presently unclear. Here, we describe expression and purification methods for recombinant CDTa and CDTb produced in Escherichia coli. We show that purified CDTa and CDTb can combine to form an active CDT which is cytotoxic to Vero cells. In addition, the purification processes described will allow milligram quantities of binary toxin fragments to be produced for further functional and structural studies.

Molecular features of the sortase enzyme family

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.

Structure and function of a Clostridium difficile sortase enzyme.

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.

Recombinant antigens based on toxins A and B of Clostridium difficile that evoke a potent toxin-neutralising immune response

Highlights • Definition of Clostridium difficile toxin-derived antigens for soluble expression in E. coli.• Demonstration of their potent neutralising immune response against key epidemic strain toxins.• TcdA and TcdB were different with respect to the domains that evoke a neutralising immune response.• TcdB central domains dominate the generation of a toxin-neutralising response.• Generated antibodies prevent C. difficile infection in passive immunisation studies.

The Structure of the Cysteine Protease and Lectin-Like Domains of Cwp84, a Surface Layer-Associated Protein from Clostridium Difficile

The crystal structure of Cwp84, an S-layer protein from Clostridium difficile is presented for the first time. The cathepsin L-like fold of cysteine protease domain, a newly observed ‘lectin-like’ domain and several other features are described.

Cwp84, a Clostridium difficile cysteine protease, exhibits conformational flexibility in the absence of its propeptide

Two structures of Cwp84, a cysteine protease from the S-layer of C. difficile, are presented after propeptide cleavage. They reveal the movement of three loops, two in the active-site groove and one on the surface of the lectin-like domain, exposing a hydrophobic pocket.

Cwp2 from Clostridium difficile exhibits an extended three domain fold and cell adhesion in vitro

Colonization of the gut by Clostridium difficile requires the adhesion of the bacterium to host cells. A range of cell surface located factors have been linked to adhesion including the S‐layer protein LMW SLP and the related protein Cwp66. As well as these proteins, the S‐layer of C. difficile may contain many others. One such protein is Cwp2. Here, we demonstrate the production of a C. difficile strain 630 cwp2 knockout mutant and assess the effect on the bacterium. The mutant results in increased TcdA (toxin A) release and impaired cellular adherence in vitro. We also present the extended three domain structure of the ‘functional’ region of Cwp2, consisting of residues 29–318 at 1.9 Å, which is compared to that of LMW SLP and Cwp8. The adhesive properties of Cwp2 and LMW SLP, which are likely to be shared by Cwp8, are predicted to be mediated by the variable loop regions in domain 2.