Structure and function of a <i>Clostridium difficile</i> 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

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

In recent decades, the global healthcare problems caused by Clostridium difficile have increased at an alarming rate. A greater understanding of this antibiotic-resistant bacterium, particularly with respect to how it interacts with the host, is required for the development of novel strategies for fighting C. difficile infections. The surface layer (S-layer) of C. difficile is likely to be of significant importance to host–pathogen interactions. The mature S-layer is formed by a proteinaceous array consisting of multiple copies of a high-molecular-weight and a low-molecular-weight S-layer protein. These components result from the cleavage of SlpA by Cwp84, a cysteine protease. The structure of a truncated Cwp84 active-site mutant has recently been reported and the key features have been identified, providing the first structural insights into the role of Cwp84 in the formation of the S-layer. Here, two structures of Cwp84 after propeptide cleavage are presented and the three conformational changes that are observed are discussed. These changes result in a reconfiguration of the active site and exposure of the hydrophobic pocket

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

AbstractInfection with the bacterium Clostridium difficile causes symptoms ranging from mild to severe diarrhoea with life-threatening complications and remains a significant burden to healthcare systems throughout the developed world. Two potent cytotoxins, TcdA and TcdB are the prime mediators of the syndrome and rapid neutralisation of these would afford significant benefits in disease management. In the present study, a broad range of non-toxic, recombinant fragments derived from TcdA and TcdB were designed for soluble expression in E. coli and assessed for their capacity to generate a potent toxin-neutralising immune response as assessed by cell-based assays. Significant differences between the efficacies of isolated TcdA and TcdB regions with respect to inducing a neutralising immune response were observed. While the C-terminal repeat regions played the principal role in generating neutralising antibodies to TcdA, in the case of TcdB, the central region domains dominated the neutralising immune response. For both TcdA and TcdB, fragments which comprised domains from both the central and C-terminal repeat region of the toxins were found to induce the most potent neutralising immune responses. Generated antibodies neutralised toxins produced by a range of C. difficile isolates including ribotype 027 and 078 strains. Passive immunisation of hamsters with a combination of antibodies to TcdA and TcdB fragments afforded complete protection from severe CDI induced by a challenge of bacterial spores. The results of the study are discussed with respect to the development of a cost effective immunotherapeutic approach for the management of C. difficile infection

High prevalence of subclass-specific binding and neutralizing antibodies against Clostridium difficile toxins in adult cystic fibrosis sera: possible mode of immunoprotection against symptomatic C. difficile infection

Objectives: Despite multiple risk factors and a high rate of colonization for Clostridium difficile, the occurrence of C. difficile infection in patients with cystic fibrosis is rare. The aim of this study was to compare the prevalence of binding C. difficile toxin-specific immunoglobulin (Ig)A, IgG and anti-toxin neutralizing antibodies in the sera of adults with cystic fibrosis, symptomatic C. difficile infection (without cystic fibrosis) and healthy controls. Methods: Subclass-specific IgA and IgG responses to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B only expressing strain (CCUG 20309) and precursor form of B fragment of binary toxin, pCDTb, were determined by protein microarray. Neutralizing antibodies to C. difficile toxins A and B were evaluated using a Caco-2 cell-based neutralization assay. Results: Serum IgA anti-toxin A and B levels and neutralizing antibodies against toxin A were significantly higher in adult cystic fibrosis patients (n=16) compared with healthy controls (n=17) and patients with symptomatic C. difficile infection (n=16); p≤0.05. The same pattern of response prevailed for IgG, except that there was no difference in anti-toxin A IgG levels between the groups. Compared with healthy controls (toxins A and B) and patients with C. difficile infection (toxin A), sera from cystic fibrosis patients exhibited significantly stronger protective anti-toxin neutralizing antibody responses. Conclusion: A superior ability to generate robust humoral immunity to C. difficile toxins in the cystic fibrosis population is likely to confer protection against symptomatic C. difficile infection. This protection may be lost in the post-transplantation setting, where sera-monitoring of anti-C. difficile toxin antibody titers may be of clinical value

A novel inhibitor prevents the peripheral neuroparalysis of Botulinum neurotoxins

Botulinum neurotoxins (BoNTs) form a large class of potent and deadly neurotoxins. Given their growing number, it is of paramount importance to discover novel inhibitors targeting common steps of their intoxication process. Recently, EGA was shown to inhibit the action of bacterial toxins and viruses exhibiting a pH-dependent translocation step in mammalian cells, by interfering with their entry route. As BoNTs act in the cytosol of nerve terminals, the entry into an appropriate compartment wherefrom they translocate the catalytic moiety is essential for toxicity. Herein we propose an optimized procedure to synthesize EGA and we show that, in vitro, it prevents the neurotoxicity of different BoNT serotypes by interfering with their trafficking. Furthermore, in mice, EGA mitigates botulism symptoms induced by BoNT/A and significantly decreases the lethality of BoNT/B and BoNT/D. This opens the possibility of using EGA as a lead compound to develop novel inhibitors of botulinum neurotoxins

Profiling humoral immune responses to Clostridium difficile-specific antigens by protein microarray analysis

Clostridium difficile is an anaerobic, Gram-positive, and spore-forming bacterium that is the leading worldwide infective cause of hospital-acquired and antibiotic-associated diarrhea. Several studies have reported associations between humoral immunity and the clinical course of C. difficile infection (CDI). Host humoral immune responses are determined using conventional enzyme-linked immunosorbent assay (ELISA) techniques. Herein, we report the first use of a novel protein microarray assay to determine systemic IgG antibody responses against a panel of highly purified C. difficile-specific antigens, including native toxins A and B (TcdA and TcdB, respectively), recombinant fragments of toxins A and B (TxA4 and TxB4, respectively), ribotypespecific surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). Microarrays were probed with sera from a total of 327 individuals with CDI, cystic fibrosis without diarrhea, and healthy controls. For all antigens, precision profiles demonstrated<10% coefficient of variation (CV). Significant correlation was observed between microarray and ELISA in the quantification of antitoxin A and antitoxin B IgG. These results indicate that microarray is a suitable assay for defining humoral immune responses to C. difficile protein antigens and may have potential advantages in throughput, convenience, and cost

Expression, purification, crystallization and preliminary crystallographic analysis of a putative Clostridium difficile surface protein Cwp19

Cwp19 is a putatively surface-located protein from Clostridium difficile. A recombinant N-terminal protein (residues 27–401) lacking the signal peptide and the C-terminal cell-wall-binding repeats (PFam04122) was crystallized using the sitting-drop vapour-diffusion method and diffracted to 2 Å resolution