Cellular stability of Rho-GTPases glucosylated by Clostridium difficile toxin B

Mono-glucosylation of Rho, Rac, and Cdc42 by Clostridium difficile toxin B (TcdB) induces changes of actin dynamics and apoptosis. When fibroblasts were treated with TcdB, an apparent decrease of the cellular Rac1 level was observed when applying anti-Rac1(Mab 102). This decrease was not based on degradation as inhibition of the proteasome by lactacystin did not stabilise cellular Rac1 levels. The application of anti-Rac1 (Mab 23A8) showed that the cellular Rac1 level slightly increased in TcdB-treated fibroblasts; thus, the apparent loss of cellular Rac1 was not due to degradation but due to impaired recognition of glucosylated Rac1 by anti-Rac1 (Mab 102). In contrast, recognition of RhoA by anti-RhoA (Mab 26C4) and Cdc42 by anti-Cdc42 (Mab 44) was not altered by glucosylation; a transient decrease of cellular RhoA and Cdc42 in TcdB-treated fibroblasts was indeed due to proteasomal degradation, as inhibition of the proteasome by lactacystin stabilised both cellular RhoA and Cdc42 levels. The finding that the apparent decrease of Rac1 reflects Rac1 glucosylation offers a valuable tool to determine Rac1 glucosylation.     

Clostridium difficile Hfq can replace Escherichia coli Hfq for most of its function

A gene for the Hfq protein is present in the majority of sequenced bacterial genomes. Its characteristic hexameric ring-like core structure is formed by the highly conserved N-terminal regions. In contrast, the C-terminal forms an extension, which varies in length, lacks homology, and is predicted to be unstructured. In Gram-negative bacteria, Hfq facilitates the pairing of sRNAs with their mRNA target and thus affects gene expression, either positively or negatively, and modulates sRNA degradation. In Gram-positive bacteria, its role is still poorly characterized. Numerous sRNAs have been detected in many Gram-positive bacteria, but it is not yet known whether these sRNAs act in association with Hfq. Compared with all other Hfqs, the C. difficile Hfq exhibits an unusual C-terminal sequence with 75% asparagine and glutamine residues, while the N-terminal core part is more conserved. To gain insight into the functionality of the C. difficile Hfq (Cd-Hfq) protein in processes regulated by sRNAs, we have tested the ability of Cd-Hfq to fulfill the functions of the E. coli Hfq (Ec-Hfq) by examining various functions associated with Hfq in both positive and negative controls of gene expression. We found that Cd-Hfq substitutes for most but not all of the tested functions of the Ec-Hfq protein. We also investigated the role of the C-terminal part of the Hfq proteins. We found that the C-terminal part of both Ec-Hfq and Cd-Hfq is not essential but contributes to some functions of both the E. coli and C. difficile chaperons.     

Chondroitin sulfate proteoglycan 4 functions as the cellular receptor for Clostridium difficile toxin B

As a gram-positive, spore-forming anaerobic bacillus, Clostridium difficile (C. difficile) is responsible for severe and fatal pseudomembranous colitis, and poses the most urgent antibiotic resistance threat worldwide. Epidemic C. difficile is the leading cause of antibiotic-associated diarrhoea globally, especially diarrhoea due to the emergence of hypervirulent strains associated with high mortality and morbidity. TcdB, one of the key virulence factors secreted by this bacterium, enters host cells through a poorly understood mechanism to elicit its pathogenic effect. Here we report the first identification of the TcdB cellular receptor, chondroitin sulfate proteoglycan 4 (CSPG4). CSPG4 was initially isolated from a whole-genome human shRNAmir library screening, and its role was confirmed by both TALEN- and CRISPR/Cas9-mediated gene knockout in human cells. CSPG4 is critical for TcdB binding to the cell surface, inducing cytoskeleton disruption and cell death. A direct interaction between the N-terminus of CSPG4 and the C-terminus of TcdB was confirmed, and the soluble peptide of the toxin-binding domain of CSPG4 could protect cells from the action of TcdB. Notably, the complete loss of CSPG4/NG2 decreased TcdB-triggered interleukin-8 induction in mice without significantly affecting animal mortality. Based on both the in vitro and in vivo studies, we propose a dual-receptor model for TcdB endocytosis. The discovery of the first TcdB receptor reveals a previously unsuspected role for CSPG4 and provides a new therapeutic target for the treatment of C. difficile infection.     

Structural determinants of Clostridium difficile toxin A glucosyltransferase activity

The principle virulence factors in Clostridium difficile pathogenesis are TcdA and TcdB, homologous glucosyltransferases capable of inactivating small GTPases within the host cell. We present crystal structures of the TcdA glucosyltransferase domain in the presence and absence of the co-substrate UDP-glucose. Although the enzymatic core is similar to that of TcdB, the proposed GTPase-binding surface differs significantly. We show that TcdA is comparable with TcdB in its modification of Rho family substrates and that, unlike TcdB, TcdA is also capable of modifying Rap family GTPases both in vitro and in cells. The glucosyltransferase activities of both toxins are reduced in the context of the holotoxin but can be restored with autoproteolytic activation and glucosyltransferase domain release. These studies highlight the importance of cellular activation in determining the array of substrates available to the toxins once delivered into the cell.     

High temporal resolution of glucosyltransferase dependent and independent effects of Clostridium difficile toxins across multiple cell types

Background

Clostridium difficile toxins A and B (TcdA and TcdB), considered to be essential for C. difficile infection, affect the morphology of several cell types with different potencies and timing. However, morphological changes over various time scales are poorly characterized. The toxins’ glucosyltransferase domains are critical to their deleterious effects, and cell responses to glucosyltransferase-independent activities are incompletely understood. By tracking morphological changes of multiple cell types to C. difficile toxins with high temporal resolution, cellular responses to TcdA, TcdB, and a glucosyltransferase-deficient TcdB (gdTcdB) are elucidated.

Results

Human umbilical vein endothelial cells, J774 macrophage-like cells, and four epithelial cell lines (HCT8, T84, CHO, and immortalized mouse cecal epithelial cells) were treated with TcdA, TcdB, gdTcdB. Impedance across cell cultures was measured to track changes in cell morphology. Metrics from impedance data, developed to quantify rapid and long-lasting responses, produced standard curves with wide dynamic ranges that defined cell line sensitivities. Except for T84 cells, all cell lines were most sensitive to TcdB. J774 macrophages stretched and increased in size in response to TcdA and TcdB but not gdTcdB. High concentrations of TcdB and gdTcdB (>10 ng/ml) greatly reduced macrophage viability. In HCT8 cells, gdTcdB did not induce a rapid cytopathic effect, yet it delayed TcdA and TcdB’s rapid effects. gdTcdB did not clearly delay TcdA or TcdB’s toxin-induced effects on macrophages.

Conclusions

Epithelial and endothelial cells have similar responses to toxins yet differ in timing and degree. Relative potencies of TcdA and TcdB in mouse epithelial cells in vitro do not correlate with potencies in vivo. TcdB requires glucosyltransferase activity to cause macrophages to spread, but cell death from high TcdB concentrations is glucosyltransferase-independent. Competition experiments with gdTcdB in epithelial cells confirm common TcdA and TcdB mechanisms, yet different responses of macrophages to TcdA and TcdB suggest different, additional mechanisms or targets in these cells. This first-time, precise quantification of the response of multiple cell lines to TcdA and TcdB provides a comparative framework for delineating the roles of different cell types and toxin-host interactions.

Electronic supplementary material

The online version of this article (doi:10.1186/s12866-015-0361-4) contains supplementary material, which is available to authorized users.     

Clostridium difficile cell wall protein CwpV undergoes enzyme-independent intramolecular autoproteolysis

Clostridium difficile infection is a leading cause of antibiotic-associated diarrhea, placing considerable economic pressure on healthcare systems and resulting in significant morbidity and mortality. The pathogen produces a proteinaceous array on its cell surface known as the S-layer, consisting primarily of the major S-layer protein SlpA and a family of SlpA homologs. CwpV is the largest member of this family and is expressed in a phase-variable manner. The protein is post-translationally processed into two fragments that form a noncovalent, heterodimeric complex. To date, no specific proteases capable of cleaving CwpV have been identified. Using site-directed mutagenesis we show that CwpV undergoes intramolecular autoproteolysis, most likely facilitated by a N-O acyl shift, with Thr-413 acting as the source of a nucleophile driving this rearrangement. We demonstrate that neighboring residues are also important for correct processing of CwpV. Based on protein structural predictions and analogy to the glycosylasparaginase family of proteins, it appears likely that these residues play key roles in determining the correct protein fold and interact directly with Thr-413 to promote nucleophilic attack. Furthermore, using a cell-free protein synthesis assay we show that CwpV maturation requires neither cofactors nor auxiliary enzymes.     

Prevention of the cytopathic effect induced by Clostridium difficile Toxin B by active Rac1

Clostridium difficile Toxin B (TcdB) glucosylates low molecular weight GTP-binding proteins of the Rho subfamily and thereby causes actin re-organization (cell rounding). This "cytopathic effect" has been generally attributed to RhoA inactivation. Here we show that cells expressing non-glucosylatable Rac1-Q61L are protected from the cytopathic effect of TcdB. In contrast, cells expressing RhoA-Q63L or mock-transfected cells are fully susceptible for the cytopathic effect of TcdB. These findings are extended to the Rac1/RhoG mimic IpgB1 and the RhoA mimic IpgB2 from Shigella. Ectopic expression of IpgB1, but not IpgB2, counteracts the cytopathic effect of TcdB. These data strongly suggest that Rac1 rather than RhoA glucosylation is critical for the cytopathic effect of TcdB.     

Clostridium difficile has an original peptidoglycan structure with a high level of N-acetylglucosamine deacetylation and mainly 3-3 cross-links

The structure of the vegetative cell wall peptidoglycan of Clostridium difficile was determined by analysis of its constituent muropeptides with a combination of reverse-phase high pressure liquid chromatography separation of muropeptides, amino acid analysis, mass spectrometry and tandem mass spectrometry. The structures assigned to 36 muropeptides evidenced several original features in C. difficile vegetative cell peptidoglycan. First, it is characterized by a strikingly high level of N-acetylglucosamine deacetylation. In addition, the majority of dimers (around 75%) contains A(2)pm(3) → A(2)pm(3) (A(2)pm, 2,6-diaminopimelic acid) cross-links and only a minority of the more classical Ala(4) → A(2)pm(3) cross-links. Moreover, a significant amount of muropeptides contains a modified tetrapeptide stem ending in Gly instead of D-Ala(4). Two L,D-transpeptidases homologues encoding genes present in the genome of C. difficile 630 and named ldt(cd1) and ldt(cd2), were inactivated. The inactivation of either ldt(cd1) or ldt(cd2) significantly decreased the abundance of 3-3 cross-links, leading to a marked decrease of peptidoglycan reticulation and demonstrating that both ldt(cd1)-and ldt(cd2)-encoded proteins have a redundant L,D-transpeptidase activity. The contribution of 3-3 cross-links to peptidoglycan synthesis increased in the presence of ampicillin, indicating that this drug does not inhibit the L,D-transpeptidation pathway in C. difficile.     

Peptide antibiotic and actin-binding protein as mixed-type inhibitors of Clostridium difficile CDT toxin activities

CDT from Clostridium difficile is an ADP-ribosyltransferase that causes rapid actin disaggregation and cell death. For efficient catalysis, CDT required specific divalent cations and binding by NAD which can be substituted by ATP but not ADP. Increasing isolation of CDT-producing strains prompted our search for antagonists like the anti-C. difficile agents bacitracin and vancomycin which were effective CDT inhibitors. Other CDT transferase and glycohydrolase inhibitors with consistently low IC50 values were heterocyclic peptide antibiotics containing modified amino acids such as polymyxin B and beta-lactam cephalosporins. The strongest inhibitors were actin-binding proteins which possess extensive interfaces with G-actin, adjoining the CDT-ADP-ribose+ acceptor site and nucleotide cleft. Analysis of the extent and mode of inhibition and actin interaction sites provided fresh evidences on the designation of actin interface domains with actin-binding proteins. Our results uphold ADP-ribosylation as an innate physiologic process in cellular cytoskeletal reorganization regulated by endogenous metabolites.     

The chemical structure and genetic locus of Campylobacter jejuni CG8486 (serotype HS:4) capsular polysaccharide: the identification of 6-deoxy-D-ido-heptopyranose

In line with our on-going efforts to create a multivalent anti-Campylobacter jejuni vaccine based on its capsule polysaccharides (CPSs), we report here the chemical structure and the genetic locus of the CPS produced by C. jejuni strain CG8486, which belongs to the serotype HS:4 CPS complex. C. jejuni CG8486 CPS was observed to be composed of approximately 17 disaccharide repeating blocks of 4-substituted N-acetyl-beta-D-glucopyranosamine and 3-substituted 6-deoxy-beta-D-ido-heptopyranose. A small number of 6-deoxy-beta-D-ido-heptopyranose units were observed to carry O-methyl phosphoramidate moieties at the O-2 or O-7 position. The gene content and organization of the CPS locus of C. jejuni CG8486 were comparable to those of C. jejuni strains NCTC 11168 and 81-176, but several CG8486 CPS genes were observed to be more divergent from those present in the CPS loci of NCTC 11168 and 81-176 CPS, which indicated that there are genetic characteristics specific to the C. jejuni HS:4 CPS complex. The efficacy of a glycoconjugate vaccine based on C. jejuni CG8486 CPS is presently being tested in an animal model, the results of which will be presented in future communications.     

Reset of a critically disturbed microbial ecosystem: faecal transplant in recurrent Clostridium difficile infection

Recurrent Clostridium difficile infection (CDI) can be effectively treated by infusion of a healthy donor faeces suspension. However, it is unclear what factors determine treatment efficacy. By using a phylogenetic microarray platform, we assessed composition, diversity and dynamics of faecal microbiota before, after and during follow-up of the transplantation from a healthy donor to different patients, to elucidate the mechanism of action of faecal infusion. Global composition and network analysis of the microbiota was performed in faecal samples from nine patients with recurrent CDI. Analyses were performed before and after duodenal donor faeces infusion, and during a follow-up of 10 weeks. The microbiota data were compared with that of the healthy donors. All patients successfully recovered. Their intestinal microbiota changed from a low-diversity diseased state, dominated by Proteobacteria and Bacilli, to a more diverse ecosystem resembling that of healthy donors, dominated by Bacteroidetes and Clostridium groups, including butyrate-producing bacteria. We identified specific multi-species networks and signature microbial groups that were either depleted or restored as a result of the treatment. The changes persisted over time. Comprehensive and deep analyses of the microbiota of patients before and after treatment exposed a therapeutic reset from a diseased state towards a healthy profile. The identification of microbial groups that constitute a niche for C. difficile overgrowth, as well as those driving the reinstallation of a healthy intestinal microbiota, could contribute to the development of biomarkers predicting recurrence and treatment outcome, identifying an optimal microbiota composition that could lead to targeted treatment strategies.     

Molecular cloning, overexpression in Escherichia coli, and purification of 6x his-tagged C-terminal domain of Clostridium difficile toxins A and B

Genomic DNA from ribotype-01 and -17 Clostridium difficile strains was used for amplification of the sequences encoding the carboxy-terminal domain of toxins A (TcdA) and B (TcdB). The deduced C-terminal TcdB ribotype-01 and -17 domains share 99.5% amino acid sequence identity while TcdA ribotype-17 comprises a 607 amino acid deletion compared to TcdA-01. When compared to previously sequenced C. difficile toxins, 99.3% amino acid identity was found between TcdA-01 and TcdA from strain VPI10643 and 98.8% identity between TcdA-17 and TcdA from strain F-1470. The obtained sequences were fused in 3' to a sequence encoding a hexahistidine tag and cloned into an Escherichia coli expression vector. The recombinant proteins were expressed in E. coli and purified using single-step metal-chelate chromatography. The recombinant carboxy-terminal domain of TcdA-01 was purified from the soluble E. coli lysate fraction whereas TcdA-17 and TcdB-17 carboxy-terminal domains were purified from inclusion bodies. At least 40 mg of each protein was purified per liter of bacterial culture. The recombinant toxin domains were detected specifically by Western blot and ELISA with antibodies against native C. difficile toxins. This study demonstrated that the carboxy-terminal domains of TcdA and TcdB can be produced using an E. coli expression system and easily purified. These recombinant, stable, and non-toxic proteins provide a convenient source for use in the diagnosis of C. difficile infections, instead of native toxins, as controls and calibrators in immunoassay kits and to obtain specific monoclonal antibodies.     

Efficient expression and purification of methyltransferase in acetyl-coenzyme a synthesis pathway of the human pathogen Clostridium difficile

The Wood-Ljungdahl pathway is responsible for acetyl-CoA biosynthesis and used as a major mean of generating energy for growth in some anaerobic microbes. Series of genes, from the anaerobic human pathogen Clostridium difficile, have been identified that show striking similarity to the genes involved in this pathway including methyltetrahydrofolate- and corrinoid-dependent methyltransferase. This methyltransferase plays a central role in this pathway that transfers the methyl group from methyltetrahydrofolate to a cob(I)amide center in the corrinoid iron-sulfur protein. In this study, we developed two efficient expression and purification methods for methyltransferase from C. difficile for the first time with two expression vectors MBPHT-mCherry2 and pETDuet-1, respectively. Using the latter vector, more than 50mg MeTr was produced per liter Luria-Bertani broth media. The recombinant methyltransferase was well characterized by SDS-PAGE, gel filtration chromatography, enzyme assay and far-UV circular dichroism (CD). Furthermore, a highly effective approach was established for determining the methyl transfer activity of the methyltetrahydrofolate- and cobalamin-dependent methyltransferase using exogenous cobalamin as a substrate by stopped-flow method. These results will provide a solid basis for further study of the methyltransferase and the Wood-Ljungdahl pathway.     

Analysis of prevalence, risk factors and molecular epidemiology of Clostridium difficile infection in Kuwait over a 3-year period

We conducted a prospective study to evaluate the prevalence and epidemiology of CDI in Kuwait government hospitals over a 3-year period, January 2003 to December 2005, to determine the ribotypes responsible for CDI and to estimate the prevalence of ribotype 027. We also conducted a case-control study to identify the risk factors in our patient population. A total of 697 stool samples from patients with suspected CDI were obtained and sent to Anaerobe Reference Laboratory, Faculty of Medicine, Kuwait University for Clostridium difficile toxin detection, culture and PCR ribotyping. During the period, 73 (10.5%) out of 697 patients met the case definition of CDI. Of these, 56 (76.7%) were hospital-acquired and 17 (23.3%) were from outpatient clinics. Thus, the prevalence of hospital-acquired CDI amongst patients with diarrhoea was 8% over the study period; the prevalence in 2003, 2004 and 2005 was 9.7%, 7.8% and 7.2%, respectively. Our data showed that 42.9% of the CDI patients were above 60 years, of which >79% were aged 71 years and above. Patients with CDI were more likely than the controls to have been exposed to immunosuppressive drugs and feeding via nasogastric tube. The most common ribotypes isolated during this study were 002, 001, 126 and 140 and they represent 55.1% of all isolates. PCR ribotype 027 was not isolated.     

Structural characterization of the cell wall binding domains of Clostridium difficile toxins A and B; evidence that Ca2+ plays a role in toxin A cell surface association

Clostridium difficile (C.difficile) is a nosocomially acquired intestinal bacillus which can cause chronic diarrhea and life-threatening colitis. The pathogenic effects of the bacillus are mediated by the release of two toxins, A and B. The C-terminal portions of both toxins are composed of 20 and 30 residue repeats known as cell wall binding (CWB) domains. We have cloned and expressed the CWB-domains of toxins A and B and several truncated CWB-domain constructs to investigate their structure and function. The smallest CWB-domain that folded in a cooperative manner was an 11 repeat construct of toxin A. This differentiates the C-terminal domains of toxins A and B from the CWB-domain of Streptococcus pneumoniae LytA, which only requires six repeats to fold. The 11 repeat toxin A construct bound Ca2+ directly with millimolar affinity and interacted with mammalian cell surfaces in a concentration and Ca2+-dependent fashion. Millimolar Ca2+ levels also accelerated toxin mediated CHO cell killing in an in vitro cell assay. Together, the data suggest a role for extracellular Ca2+ in the sensitization of toxin A/cell-surface interactions.     

Non contiguous-finished genome sequence and description of Clostridium jeddahense sp. nov.

Clostridium jeddahense strain JCD^T (= CSUR P693 = DSM 27834) is the type strain of C. jeddahense sp. nov. This strain, whose genome is described here, was isolated from the fecal flora of an obese 24 year-old Saudian male (BMI=52 kg/m²). Clostridium jeddahense strain JCD^T is an obligate Gram-positive bacillus. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 3,613,503 bp long genome (1 chromosome, no plasmid) exhibits a G+C content of 51.95% and contains 3,462 protein-coding and 53 RNA genes, including 4 rRNA genes.     

Four Distinct Structural Domains in Clostridium difficile Toxin B Visualized Using SAXS

Clostridium difficile is a nosocomial bacterial pathogen causing antibiotic-associated diarrhea and fatal pseudomembranous colitis. Key virulence factors are toxin A and toxin B (TcdB), two highly related toxins that are members of the large clostridial toxin family. These large multifunctional proteins disrupt cell function using a glucosyltransferase domain that is translocated into the cytosol after vesicular internalization of intact holotoxin. Although substantial information about the biochemical mechanisms of intoxication exists, research has been hampered by limited structural information, particularly of intact holotoxin. Here, we used small-angle X-ray scattering (SAXS) methods to obtain an ab initio low-resolution structure of native TcdB, which demonstrated that this molecule is monomeric in solution and possesses a highly asymmetric shape with a maximum dimension of ∼ 275 Å. Combining this SAXS information with crystallographic or modeled structures of individual functional domains of TcdB reveals for the first time that the three-dimensional structure of TcdB is organized into four distinct structural domains. Structures of the N-terminal glucosyltransferase, the cysteine protease, and the C-terminal repeat region can be aligned within three domains of the SAXS envelope. A fourth domain, predicted to be involved in the translocation of the glucosyltransferase, appears as a large solvent-exposed protrusion. Knowledge of the shapes and relative orientations of toxin domains provides new insight into defining functional domain boundaries and provides a framework for understanding how potential intra-domain interactions enable conformational changes to propagate between domains to facilitate intoxication processes.     

Identification and modeling of a drug target for Clostridium perfringens SM101

In the present study, comparative genome analysis between Clostridium perfringens and the human genome was carried out to identify genes that are essential for the pathogen's survival, and non-homologous to the genes of human host, that can be used as potential drug targets. The study resulted in the identification of 426 such genes. The number of these potential drug targets thus identified is significantly lower than the genome's protein coding capacity (2558 protein coding genes). The 426 genes of C. perfringens were further analyzed for overall similarities with the essential genes of 14 different bacterial species present in Database of Essential Genes (DEG). Our results show that there are only 5 essential genes of C. perfringens that exhibit similarity with 12 species of the 14 different bacterial species present in DEG database. Of these, 1 gene was similar in 12 species and 4 genes were similar in 11 species. Thus, the study opens a new avenue for the development of potential drugs against the highly pathogenic bacterium. Further, by selecting these essential genes of C. perfringens, which are common and essential for other pathogenic microbial species, a broad spectrum anti-microbial drug can be developed. As a case study, we have built a homology model of one of the potential drug targets, ABC transporter-ATP binding protein, which can be employed for in silico docking studies by suitable inhibitors.