Profile of toxigenicity of Clostridium difficile strains isolated from paediatric patients with clinical diagnosis of antibiotic associated diarrhea (AAD)

This study was performed to determine profile of toxigenicity of 18 Clostridium difficile strains isolated from paeditric patients suffering from antibiotic associated diarrhea (AAD). Toxigenicity of C. difficile strains was tested for detection toxin A and toxin B by phenotypic methods and for detection of the tcdA and tcdB genes using of PCR. Changes in the repeating regions of the tcdA genes were detected with the NK9/NKV011 primer pairs. For detection of binary toxin (CDT) cdtA and cdtB genes, cdtApos/cdtArev i cdtBpos/cdtBrev two pair primers in PCR was used. Among C. difficile strains was detected three profiles of toxigenicity: C. difficile strains possesing of tcdA and tcdB genes but not possesing cdtA and cdtB genes of binary toxin (A+B+CDT-), strains possesing tcdA and tcdB and cdtA and cdtB genes (A+B+CDT+), strains with deletion of toxin A gene (A-B+CDT-). This is the first report on the occurence of binary positive C. difficile strains isolated from paediatric patients.     

Evaluation of different methods for detection of Clostridium difficile toxins in Poland

The aim of this study was to compare different methods for C. difficile toxins detection. Fifty three stool samples taken from patients with antibiotic-associated diarrhoea were studied. TCD toxin A EIA (Becton Dickinson, USA), Tox A/B ELISA test (TechLab, USA), cytotoxicity and neutralization assay on McCoy cells and PCR for detection of both toxin A and B genes were performed in vivo (in stool samples) and in vitro (in isolated strains). Reference toxigenic and nontoxigenic and two Japanese toxin A-negative and toxin B-positive C. difficile strains were used as a controls. TCD toxin A EIA detected in vivo only 19 positive samples. Tox A/B test detected 52 positive samples out of 53 studied. All 53 stool samples were C. difficile culture positive (53 strains were cultured). Toxin B was detected in 52 strain-supernatants and in all controls (except the nontoxigenic one). Both toxin A and B genes were detected by PCR in all 53 isolated strains, Japanese and reference strain (except the nontoxigenic one). In vitro toxin A was detected by TCD toxin A EIA in 42 strains. These results were compared with those obtained in Tox A/B ELISA test. We observed 52 positive strains. Toxigenic reference strain and two Japanese toxA(-)/toxB(+) strains were also positive. Only 2 negative results were obtained with the nontoxigenic reference strain and unique nontoxigenic isolated strain. Tox A/B ELISA test seems to be the best for detection of C. difficile toxins in vivo and in vitro. Test avoids the false-negative results in the case of presence of toxin A-negative and toxin B-positive strain.     

Inhibition of protein tyrosine phosphatases suppresses P-selectin exocytosis in activated human platelets

The cytolethal distending toxin (CDT) of Haemophilus ducreyi is encoded by the cdtABC genes, but the composition of active CDT is not known. Both immunoaffinity and metal affinity chromatographic methods were used to purify H. ducreyi CDT from recombinant Escherichia coli strains bearing wild-type or mutated H. ducreyi cdtABC genes. Both affinity-purified preparations contained CdtA, CdtB, and CdtC proteins. These purification efforts also revealed that the formation of a noncovalent CdtB-CdtC complex and production of a fully active CDT complex required the presence of a functional CdtA protein. When purified recombinant CdtB and CdtC proteins were mixed, only very slight CDT activity was detected. In contrast, when a bacterial cell extract containing CdtA was mixed with purified preparations of both CdtB and CdtC, full CDT activity was reconstituted in vitro. These results indicate that CdtA is essential for normal H. ducreyi CDT activity and that CdtA likely modifies or alters either CdtB or CdtC or both to form the active CDT complex.     

Characterization of Putative Cholesterol Recognition/Interaction Amino Acid Consensus-Like Motif of Campylobacter jejuni Cytolethal Distending Toxin C

Cytolethal distending toxin (CDT) produced by Campylobacter jejuni comprises a heterotrimeric complex formed by CdtA, CdtB, and CdtC. Among these toxin subunits, CdtA and CdtC function as essential proteins that mediate toxin binding to cytoplasmic membranes followed by delivery of CdtB into the nucleus. The binding of CdtA/CdtC to the cell surface is mediated by cholesterol, a major component in lipid rafts. Although the putative cholesterol recognition/interaction amino acid consensus (CRAC) domain of CDT has been reported from several bacterial pathogens, the protein regions contributing to CDT binding to cholesterol in C. jejuni remain unclear. Here, we selected a potential CRAC-like region present in the CdtC from C. jejuni for analysis. Molecular modeling showed that the predicted functional domain had the shape of a hydrophobic groove, facilitating cholesterol localization to this domain. Mutation of a tyrosine residue in the CRAC-like region decreased direct binding of CdtC to cholesterol rather than toxin intermolecular interactions and led to impaired CDT intoxication. These results provide a molecular link between C. jejuni CdtC and membrane-lipid rafts through the CRAC-like region, which contributes to toxin recognition and interaction with cholesterol.     

Reconstitution and purification of cytolethal distending toxin of Actinobacillus actinomycetemcomitans

Cytolethal distending toxin (CDT) has been found in various pathogenic bacterial species and causes a cell distending and a G2 arrest against eukaryotic cells. All the cdtABC genes, which encode CDT, are known to be required for the CDT activities although the CDT holotoxin structure has not been elucidated. We cloned the cdtABC genes of Actinobacillus actinomycetemcomitans and constructed an Escherichia coli expression system for them. We found that crude extracts from six deletion mutants (delta cdtA, delta cdtB, delta cdtC, delta cdtBC, delta cdtAC, and delta cdtAB) of recombinant E. coli, which showed very weak or no detectable CDT activities, restored the CDT activities when pre-mixing and pre-incubation of them were performed in combinations to contain all the CdtA, CdtB, and CdtC proteins. These results indicate that all the Cdt proteins are required for the CDT activities. We also found that the chimera CdtB protein, CdtB-intein-CBD (chitin binding domain) like CdtB protein itself assembled with CdtA and CdtC. The reconstituted CDT containing the chimera CdtB protein was specifically extracted by chitin beads and the only CDT portion was isolated from the chitin beads by a cleavage reaction of the intein. The purified reconstituted-CDT was found to consist of CdtA, CdtB, and CdtC proteins, and showed appreciable CDT activities, indicating that the CDT holotoxin structure is the CdtABC complex. To our knowledge, this is the first report succeeded in complete purification of an active CDT and may offer useful tools for elucidation of the toxic mechanism of CDT.     

beta-Helix is a likely core structure of yeast prion Sup35 amyloid fibers

Helicobacter hepaticus, a causal agent of hepatocarcinoma in mice, exhibits a cytolethal distending toxin activity. The three subunits of this holotoxin, CdtA, CdtB, and CdtC, and three CdtB mutants were produced as recombinant histidine-tagged proteins by using an in vitro cell-free protein expression system. We found that the presence of the three H. hepaticus Cdt subunits is required for cellular toxicity and that only a C-terminal CdtB mutation abolishes the activity of the complex. In vitro, H. hepaticus CdtB exhibits a DNase activity which is also abolished by this C-terminal CdtB mutation. These results suggest that the effect of H. hepaticus CDT probably involves the DNase activity of CdtB.     

Deletion and purification studies to elucidate the structure of the Actinobacillus actinomycetemcomitans cytolethal distending toxin

Cytolethal distending toxin (CDT) is one of the exotoxins produced by Actinobacillus actinomycetemcomitans, an agent of localized aggressive periodontitis. We constructed N-terminal deletion mutants of CdtA using an Escherichia coli expression system and found that ADelta19-47, with a deletion from Asn-19 to Pro-47, showed comparable CDT activity but no apparent heterogeneity of CdtA. The wild-type CDT (wtCDT) and the mutant CDT (ADelta19-47CDT) were purified to homogeneity by introducing a histidine tag into the C-terminal end of CdtB. Both purified wtCDT and purified ADelta19-47CDT showed strong CDT activity and a tripartite structure composed of CdtA (subunit A), 31 kDa CdtB (subunit B), and 18.5 kDa CdtC (subunit C) in nearly a 1:1:1 stoichiometry. Importantly, subunit A was identified as heterogeneous with three CdtA variants in wtCDT, but homogeneous in ADelta19-47CDT. Purified CDTs also showed high stability that was absolutely dependent on the presence of sucrose in the buffer. In conclusion, the region from the Asn-19 to Pro-47 of CdtA contributes to the heterogeneous production of CdtA, but is dispensable for the toxin activity. Furthermore, this study describes an effective protocol for the purification of a native rather than reconstituted CDT, and clarifies the subunit composition of the active CDT holotoxin.     

Binary toxin production in Clostridium difficile is regulated by CdtR, a LytTR family response regulator

Clostridium difficile binary toxin (CDT) is an actin-specific ADP-ribosyltransferase that is produced by various C. difficile isolates, including the "hypervirulent" NAP1/027 epidemic strains. In contrast to the two major toxins from C. difficile, toxin A and toxin B, little is known about the role of CDT in virulence or how C. difficile regulates its production. In this study we have shown that in addition to the cdtA and cdtB toxin structural genes, a functional cdt locus contains a third gene, here designated cdtR, which is predicted to encode a response regulator. By introducing functional binary toxin genes into cdtR(+) and cdtR-negative strains of C. difficile, it was established that the CdtR protein was required for optimal expression of binary toxin. Significantly increased expression of functional binary toxin was observed in the presence of a functional cdtR gene; an internal deletion within cdtR resulted in a reduction in binary toxin production to basal levels. Strains that did not carry intact cdtAB genes or cdtAB pseudogenes also did not have cdtR, with the entire cdt locus, or CdtLoc, being replaced by a conserved 68-bp sequence. These studies have shown for the first time that binary toxin production is subject to strict regulatory control by the response regulator CdtR, which is a member of the LytTR family of response regulators and is related to the AgrA protein from Staphylococcus aureus.     

Cytolethal distending toxin: limited damage as a strategy to modulate cellular functions

The coevolution of bacterial pathogens and their hosts has contributed to the development of very complex and sophisticated functional pathogen--host interfaces. Thus, well-adapted pathogens have evolved a variety of strategies to manipulate host cell functions precisely. For example, a group of unrelated Gram-negative pathogenic bacteria have evolved a toxin, known as cytolethal distending toxin (CDT), that has the ability to control cell cycle progression in eukaryotic cells. Recent studies have identified CdtB as the active subunit of the CDT holotoxin. Through its nuclease activity, CdtB causes limited DNA damage, thereby triggering the DNA-damage response that ultimately results in the observed arrest of the cell cycle. In addition, it has been established that CDT is a tripartite AB toxin in which CdtB is the active 'A' subunit and CdtA and CdtC constitute the heterodimeric 'B' subunit required for the delivery of CdtB into the target cell. The mechanism of action of CDT suggests that the infliction of limited damage could be a strategy used by pathogenic bacteria to modulate host cell functions.     

Secretome analysis of Clostridium difficile strains

Clostridium difficile causes infections ranging from mild C. difficile-associated diarrhea to severe pseudomembranous colitis. Since 2003 new hypervirulent C. difficile strains (PCR ribotype 027) emerged characterized by a dramatically increased mortality. The secretomes of the three C. difficile strains CDR20291, CD196, and CD630 were analyzed and compared. Proteins were separated and analyzed by means of SDS--PAGE and LC-MS. MS data were analyzed using Mascot and proteins were checked for export signals with SecretomeP and SignalP. LC-MS analysis revealed 158 different proteins in the supernatant of C. difficile. Most of the identified proteins originate from the cytoplasm. Thirty-two proteins in CDR20291, 36 in CD196 and 26 in CD630 were identified to be secreted by C. difficile strains. Those were mainly S-layer proteins, substrate-binding proteins of ABC-transporters, cell wall hydrolases, pilin and unknown hypothetical proteins. Toxin A and toxin B were identified after growth in brain heart infusion medium using immunological techniques. The ADP-ribosyltransferase-binding component protein, which is a part of the binary toxin CDT, was only identified in the hypervirulent ribotype 027 strains. Further proteins that are secreted specifically by hypervirulent strains were identified.     

Dynamics and assembly of the cytolethal distending toxin

The cytolethal distending toxin (CDT) is a widespread bacterial toxin that consists of an active subunit CdtB with nuclease activity and two ricin-like lectin domains, CdtA and CdtC, that are involved in the delivery of CdtB into the host cell. The three subunits form a tripartite complex that is required to achieve the fully active holotoxin. In the present study we investigate the assembly and dynamic properties of the CDT holotoxin using molecular dynamics simulations and binding free energy calculations. The results have revealed that CdtB likely adopts a different conformation in the unbound state with a closed DNA binding site. The two characterized structural elements of the aromatic patch and groove on the CdtA and CdtC protein surfaces exhibit high mobility, and free energy calculations show that the heterodimeric complex CdtA-CdtC, as well as the CdtA-CdtB and CdtB-CdtC sub-complexes are less energetically stable as compared to the binding in the tripartite complex. Analysis of the dynamical cross-correlation map reveals information on the correlated motions and long-range interplay among the CDT subunits associated with complex formation. Finally, the estimated binding free energies of subunit interactions are presented, together with the free energy decomposition to determine the contributions of residues for both binding partners, providing insight into the protein-protein interactions in the CDT holotoxin.     

In vivo virulence properties of bacterial cytolethal-distending toxin

Multiple pathogenic Gram-negative bacteria produce cytolethal-distending toxins (CDTs). CDT is typically composed of three subunits: the catalytic subunit CdtB has DNase I-like activity, whereas CdtA and CdtC are binding proteins for delivering CdtB into target cells. Translocation of CdtB to the nucleus induces genotoxic effects on host DNA, triggering DNA repair cascades that lead to cell cycle arrest and eventual cell death. Several lines of evidence indicate that this toxin contributes to the pathogenicity of CDT-producing bacteria in vivo . Helicobacter hepaticus and Campylobacter jejuni CDTs are essential for persistent infection of the gastrointestinal tract and increase the severity of mucosal inflammation or liver disease in susceptible mouse strains. Haemophilus ducreyi CDT may contribute to the pathogenesis of chancroid in rabbits. Recently, H. hepaticus CDT has been shown to play a crucial role in promoting the progression of infectious hepatitis to pre-malignant, dysplastic lesions via activation of a pro-inflammatory NF-κB pathway and increased proliferation of hepatocytes, providing the first evidence that CDT has carcinogenic potential in vivo . Thus, both in vitro and in vivo data indicate that CDT is a bacterial virulence factor.     

Detection of Clostridium difficile toxin A by reversed passive latex agglutination.

A reversed passive latex agglutination (RPLA) assay for detecting Clostridium difficile toxin A is presented. Purified monoclonal antibody (mAb 37B5) was used for latex sensitization. The culture supernatants of 93 strains of C. difficile were tested by RPLA assay and the results compared with those of a commercially available latex agglutination test, PCR and cytotoxin assay with Vero cells. There was agreement between RPLA, cytotoxicity and PCR assays, but 29 strains were positive in the RPLA assay while 35 were positive in the cytotoxicity test and PCR using primer pair NK3-NK2 directed to the nonrepeating portion of the C. difficile toxin A gene. The 6 cytotoxic but RPLA-negative strains were demonstrated to be toxin A-negative/toxin B-positive strains in the PCR assay by using primer pair NK11-NK9 directed to the repeating portion of the C. difficile toxin A gene. There were no cross-reactions with culture supernatants of the other clostridial strains except for two strains of C. sordelli that produced hemorrhagic toxin (which is immunologically related to C. difficile toxin A).     

Binary toxin producing Clostridium difficile strains

Clostridium difficile produces three toxins, TcdA, TcdB and CDT. TcdA and TcdB are single-stranded molecules acting as glucosyltransferases specific for small GTPases. CDT is an actin specific ADP-ribosylating binary toxin characteristically composed of two independent components, enzymatic CDTa (48 kDa) and binding CDTb (99 kDa). The cdtA and cdtB genes were sequenced in two CDT-positive strains of C. difficile (CD 196 and 8864) and at least two CDT-negative strains with truncated form of binary toxin genes are known (VPI 10463 and C. difficile genome strain 630). The prevalence of binary toxin producing strains is estimated to be from 1.6% to 5.5%, although a much higher proportion has been reported in some studies. The role of the binary toxin as an additional virulence factor is discussed.     

Clostridium difficile strains not producing Toxin A,but producing Toxin B [toxA(-)tox B(+)]--etiologic agent of antibiotic associated diarrhoea (AAD) in Poland

Previously, toxin A-negative/toxin B-positive Clostridium difficile strains were not thought to be associated with clinically significant diseases. In our study among 159 tested C. difficile strains isolated from feacal samples from 413 patients with antibiotic associated diarrhoea (AAD) 17 strains (11%) were negative in the "Culturette Brand Toxin" CD (Becton-Dickinson) for detection toxin A and positive in the TOX A/B test, designed for detection of both toxins. The conserved regions of both toxin genes were detectable in all of isolates studied by the PCR. Nine of these C. difficile strains had a deletion in the A gene and remaining 8 strains, revealed an amplicon with the expected size of approximately 2500 bp. In this paper we described the first time the toxin A-negative/toxin B-positive C. difficile strains with deletion in toxin A gene, isolated from the faecal samples of patient with AAD in Poland.     

Nuclear localization of the Escherichia coli cytolethal distending toxin CdtB subunit

Cytolethal distending toxin (CDT) is a heterotrimeric protein toxin produced by several bacterial pathogens. Cells exposed to CDT die from either activation of the mitotic checkpoint cascade or apoptosis. Introduction of the purified CdtB subunit, a homologue of mammalian type I DNase, into cells mimics the action of the CDT holotoxin. Mutant CdtBs lacking DNase activity are devoid of biological activity. Chromosomal DNA appears to be the CDT target; thus, nuclear translocation of CdtB must precede cytolethal activity. Examination of the CdtB sequence indicates the presence of putative candidate bipartite nuclear localization signals (NLS). Here, we examine the functionality of the two potential NLS sequences found in the Escherichia coli CdtB-II. Nuclear translocation of EcCdtB-II was examined by monitoring the localization of an EcCdtB-II-EGFP fusion in Cos-7 cells. Our results indicated that EGFP-EcCdtB-II localized to the nucleus. The candidate EcCdtB-II-II NLS sequences were modified by site-directed mutagenesis such that tandem arginine residues were changed to threonine and serine respectively. Mutation of both putative NLS sequences had no effect on EcCdtB-II-associated DNase activity; however, cell cycle arrest and nuclear localization were significantly impaired in cells that received CDT reconstituted from the EcCdtB-II-DeltaNLS mutants. When HeLa cells were electroporated with the EcCdtB-II-DeltaNLS1 and the EcCdtB-II-NLS double mutants, toxicity was not observed, whereas the activity of EcCdtB-II-DeltaNLS2 was similar to that of wild-type EcCdtB-II. These data indicate that the putative NLS sequences are important for CDT-mediated action arrest and that they are likely to function in the nuclear translocation of EcCdtB-II.     

Toxigenic Clostridium difficile PCR ribotypes from wastewater treatment plants in southern Switzerland

The occurrence of Clostridium difficile in nine wastewater treatment plants in the Ticino Canton (southern Switzerland) was investigated. The samples were collected from raw sewage influents and from treated effluents. Forty-seven out of 55 characterized C. difficile strains belonged to 13 different reference PCR ribotypes (009, 010, 014, 015, 039, 052, 053, 066, 070, 078, 101, 106, and 117), whereas 8 strains did not match any of those available in our libraries. The most frequently isolated ribotype (40%) was 078, isolated from six wastewater treatment plants, whereas ribotype 066, a toxigenic emerging ribotype isolated from patients admitted to hospitals in Europe and Switzerland, was isolated from the outgoing effluent of one plant. The majority of the isolates (85%) were toxigenic. Forty-nine percent of them produced toxin A, toxin B, and the binary toxin (toxigenic profile A(+) B(+) CDT(+)), whereas 51% showed the profile A(+) B(+) CDT(-). Interestingly, eight ribotypes (010, 014, 015, 039, 066, 078, 101, and 106) were among the riboprofiles isolated from symptomatic patients admitted to the hospitals of the Ticino Canton in 2010. Despite the limitation of sampling, this study highlights that toxigenic ribotypes of C. difficile involved in human infections may occur in both incoming and outgoing biological wastewater treatment plants. Such a finding raises concern about the possible contamination of water bodies that receive wastewater treatment plant effluents and about the safe reuse of treated wastewater.     

Functional studies of the recombinant subunits of a cytolethal distending holotoxin

Cytolethal distending toxin (CDT) is a multicomponent bacterial holotoxin that targets most eukarytotic cells causing distension and cell cycle arrest. A number of diverse pathogenic bacterial species associated with diarrhoea, chancroid, chronic hepatitis and periodontal disease produce a CDT. Synthesis of the holotoxin is directed by the expression of three genes, cdtA , cdtB and cdtC . Although the product of the CdtB gene was previously identified as a type I deoxyribonuclease, the functions of the cdtA and cdtC products have not been characterized. Using the periodontal pathogen, Actinobacillus actinomycetemcomitans , we demonstrate that the recombinant product of the CdtA gene binds to the surface of Chinese hamster ovary (CHO) cells. This protein did not induce distension or cytotoxicity when introduced into the cytosol using a lipid-based protein delivery system. Recombinant CdtB and CdtC proteins failed to bind to CHO cells. However, the delivery of either CdtB or CdtC into the cytosol resulted in the characteristic pattern of distension followed by cell death. Based on these results, it appears that the CdtA protein subunit alone is responsible for anchoring the holotoxin to the cell surface. The CdtC subunit, in concert with CdtB, contributes to the cytotoxic activities of the holotoxin. The specific mechanism of CdtC cytotoxicity is currently unknown.     

FK506-binding protein 51 interacts with Clostridium botulinum C2 toxin and FK506 inhibits membrane translocation of the toxin in mammalian cells

The binary Clostridium botulinum C2 toxin consists of the binding/translocation component C2IIa and the separate enzyme component C2I. C2IIa delivers C2I into the cytosol of eukaryotic target cells where C2I ADP-ribosylates actin. After receptor-mediated endocytosis of the C2IIa/C2I complex, C2IIa forms pores in membranes of acidified early endosomes and unfolded C2I translocates through the pores into the cytosol. Membrane translocation of C2I is facilitated by the activities of host cell chaperone Hsp90 and the peptidyl-prolyl cis/trans isomerase (PPIase) cyclophilin A. Here, we demonstrated that Hsp90 co-precipitates with C2I from lysates of C2 toxin-treated cells and identified the FK506-binding protein (FKBP) 51 as a novel interaction partner of C2I in vitro and in intact mammalian cells. Prompted by this finding, we used the specific pharmacological inhibitor FK506 to investigate whether the PPIase activity of FKBPs plays a role during membrane translocation of C2 toxin. Treatment of cells with FK506 protected cultured cells from intoxication with C2 toxin. Moreover, FK506 inhibited the pH-dependent translocation of C2I across membranes into the cytosol but did not interfere with the enzyme activity of C2I or binding of C2 toxin to cells. Furthermore, FK506 treatment delayed intoxication with the related binary actin ADP-ribosylating toxins from Clostridium perfringens (iota toxin) and Clostridium difficile (CDT) but not with the Rho-glucosylating Clostridium difficile toxin A (TcdA). In conclusion, our results support the hypothesis that clostridial binary actin-ADP-ribosylating toxins share a specific FKBP-dependent translocation mechanism during their uptake into mammalian cells.     

Isolation of toxigenic strains of Clostridium difficile and enterotoxin producing Bacteroides fragilis from fecal specimens of patients suspected of antibiotic associated diarrhoea

Fifty faecal samples from patients suspected of AAD (antibiotic associated diarrhoea) were studied for Clostridium difficile and enterotoxin producing Bacteroides fragilis (ETBF). Using TCD (Becton-Dickinson) and C. difficile Toxin A test (Oxoid) in 34% of specimens the presence of toxin A was detected. From all specimens 25 C. difficile strains were isolated. All isolated strains produced toxin B in vitro which was shown in Mc Coy cytotoxicity test. Eighteen strains only were toxin A positive in vitro. From all isolated C. difficile strains 28% were tox A (-) tox B (+). By means of PCR presence of toxin A and toxin B genes was tested directly in faecal samples and in strains. From the same 50 faecal samples 17 B. fragilis strains were isolated. Four of them produced the enterotoxin (fragilisin) which was detected on the HT 29/C1 cell line. Genes of fragilisin were found in strains and directly in faecal samples. Toxin producing C. difficile and B. fragilis (ETBF) together were found in 3 samples. From one faecal sample only ETBF was cultured.