Clostridium difficile binary toxin translocase CDTb tetradecamer in symmetric and asymmetric conformations
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ABSTRACT:
SUBMITTER: Amédée des Georges
PROVIDER: EMPIAR-11703 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:Clostridium difficile is the most common hospital acquired pathogen in the USA, and infection is, in many cases, fatal. Toxins A and B are its major virulence factors, but expression of a third toxin, known as C. difficile transferase (CDT), is increasingly common. An adenosine diphosphate (ADP)-ribosyltransferase that causes actin cytoskeletal disruption, CDT is typically produced by the major, hypervirulent strains and has been associated with more severe disease. Here, we show that CDT enhances the virulence of two PCR-ribotype 027 strains in mice. The toxin induces pathogenic host inflammation via a Toll-like receptor 2 (TLR2)-dependent pathway, resulting in the suppression of a protective host eosinophilic response. Finally, we show that restoration of TLR2-deficient eosinophils is sufficient for protection from a strain producing CDT. These findings offer an explanation for the enhanced virulence of CDT-expressing C. difficile and demonstrate a mechanism by which this binary toxin subverts the host immune response.
| S-EPMC5010011 | biostudies-literature
Project description:Clinically relevant Clostridium difficile strains usually produce toxins A and B. Some C. difficile strains can produce an additional binary toxin. We report clonality among five strains carrying all toxin genes from Polish patients with C. difficile-associated diarrhea. In another strain, possible recombination between binary toxin genes is documented.
| S-EPMC540144 | biostudies-literature
Project description:Hypervirulent epidemic strains of Clostridium difficile (C. difficile), referred to as NAPI/027, express an additional virulence factor, binary toxin (CDT), and are associated with more severe disease. Emerging evidence indicates gut immunity to C. difficile is a delicate balance between protection and pathology. To identify potential therapeutic host immune targets, we conducted a transcriptome analysis of host genes altered by NAPI/027 infection and identified interleukin-33 (IL-33) as a candidate immune target. Using a murine model, we show that both endogenous IL-33 and exogenous IL-33 treatment protect from the enhanced mortality, weight-loss and tissue pathology which is characteristic of hypervirulent C. difficile. IL-33 mediated protection was elicited through type-2 innate lymphoid cells (ILC2s) and adoptive transfer of purified ILC2s was sufficient to mitigate CDI- associated mortality and weight-loss. Furthermore, dysregulated IL-33 signaling via the soluble IL-33 decoy receptor (sST2) predicted disease severity and mortality in human patients. Lastly, colonic IL-33 expression appears to be regulated by the microbiota as antibiotic- depletion of IL-33 was rescued with mouse fecal microbiota transplant (FMT) and a human fecal spore preparation (HSP). Thus, IL-33 signaling is a novel therapeutic pathway for severe CDI which can potentially be targeted with rationally designed microbial therapies.
2019-05-09 | GSE122013 | GEO
Project description:The binary toxin Clostridium difficile transferase (CDT) is frequently observed in C. difficile strains and is associated with an increased severity of C. difficile infection. CDT-producing C. difficile infections cause higher fatality rates than infections with CDT negative isolates. Thus, the rapid and accurate identification of a CDT positive C. difficile infection is critical for effective treatment. The present study demonstrates how loop-mediated isothermal amplification (LAMP) can be used to detect CDT-producing C. difficile based on visual observation. This is a low complexity, rapid molecular method that has the potential to be used within a point of care setting. The specificity and sensitivity of the primers in the LAMP reactions for CDT detection were determined using two different methods, a real-time turbidity monitor and visual detection after the addition of calcein to the reaction tube. The results revealed that target DNA was amplified and visualized by these two detection methods within 60 min at a temperature of 60°C. The sensitivity of the LAMP assay was identified to be 10-fold greater than that of polymerase chain reaction analysis. When 25 alternative bacterial strains lacking CDT were tested, the results of the amplification were negative, confirming the specificity of the primers. In conclusion, the visual LAMP method established in the present study may be a rapid, reliable and cost-effective tool for detecting CDT-producing C. difficile strains at the point of care.
| S-EPMC5704324 | biostudies-literature
Project description:A Clostridium difficile isolate was found to produce an actin-specific ADP-ribosyltransferase (CDT) homologous to the enzymatic components of Clostridium perfringens iota toxin and Clostridium spiroforme toxin (M. R. Popoff, E. J. Rubin, D. M. Gill, and P. Boquet, Infect. Immun. 56:2299-2306, 1988). The CDT locus from C. difficile CD196 was cloned and sequenced. It contained two genes (cdtA and cdtB) which display organizations and sequences similar to those of the iota toxin gene. The deduced enzymatic (CDTa) and binding (CDTb) components have 81 and 84% identity, respectively, with the corresponding components of iota toxin. CDTa and CDTb induced actin cytoskeleton alterations similar to those caused by other clostridial binary toxins. The lower level of production of binary toxin by CD196 than of iota toxin by C. perfringens was related to a lower transcript level, possibly due to a promoter region different from that of iota toxin genes. The cdtA and cdtB genes have been detected in 3 of 24 clinical isolates examined, and cdtB alone was found in 2 additional strains. One strain (in addition to CD196) was shown by Western blotting to produce CDTa and CDTb. These results indicate that some C. difficile strains synthesize a binary toxin that could be an additional virulence factor.
| S-EPMC175146 | biostudies-other
Project description: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.
| S-EPMC2168464 | biostudies-literature
Project description:Pathogenesis of Clostridium difficile has been linked to production of toxins, including the large toxins A and B as well as the binary toxin CDT. Until recently, toxin A was only found in combination in clinical strains with the toxin B, unlike toxin B or CDT, which were found alone in toxigenic variants. New toxigenic variants of C. difficile detected in our laboratory from patients with diarrhoea or severe colitis, including a variant producing only toxin A, were tested for virulence in the hamster model, which displays the clinical features of C. difficile disease. Hamsters infected with a strain producing only toxin B induced similar clinical signs, time to death from infection and histologic damage compared to the hypervirulent strain 027. No mortality or clinical signs of infection but caecal histologic damage was found with the variant producing only toxin A. The C. difficile variant strain producing only CDT was able to kill one hamster out of seven; nevertheless, the surviving animals had few alteration of the caecum.
| S-EPMC6734109 | biostudies-literature
Project description:Clostridium difficile binary toxin (CDT) is an ADP-ribosyltransferase which is linked to enhanced pathogenesis of C. difficile strains. CDT has dual function: domain a (CDTa) catalyses the ADP-ribosylation of actin (enzymatic component), whereas domain b (CDTb) transports CDTa into the cytosol (transport component). Understanding the molecular mechanism of CDT is necessary to assess its role in C. difficile infection. Identifying amino acids that are essential to CDTa function may aid drug inhibitor design to control the severity of C. difficile infections. Here we report mutations of key catalytic residues within CDTa and their effect on CDT cytotoxicity. Rather than an all-or-nothing response, activity of CDTa mutants vary with the type of amino acid substitution; S345A retains cytotoxicity whereas S345Y was sufficient to render CDT non-cytotoxic. Thus CDTa cytotoxicity levels are directly linked to ADP-ribosyltransferase activity.
| S-EPMC5613739 | biostudies-literature
Project description:Clostridium difficile consists of six clades but studies on Clade 3 are limited. Here, we report genome sequences of three Clade 3 C. difficile strains carrying genes encoding toxin A and B and the binary toxin. Isolates 103 and 133 (both of ST5) and isolate 106 (ST285) were recovered from three ICU patients. Whole genome sequencing using HiSeq 2500 revealed 4.1-Mb genomes with 28-29% GC content. There were ≥1,104 SNP between the isolates, suggesting they were not of a single clone. The toxin A and B gene-carrying pathogenicity locus (PaLoc) of the three isolates were identical and had the insertion of the transposon Tn6218. The genetic components of PaLoc among Clade 3 strains were the same with only a few nucleotide mutations and deletions/insertions, suggesting that the Tn6218 insertion might have occurred before the divergence within Clade 3. The binary toxin-genes carrying CDT locus (CdtLoc) of the three isolates were identical and were highly similar to those of other Clade 3 strains, but were more divergent from those of other clades. In conclusion, Clade 3 has an unusual clade-specific PaLoc characteristic of a Tn6218 insertion which appears to be the main feature to distinguish Clade 3 from other C. difficile.
| S-EPMC5337907 | biostudies-literature
Project description:Clostridium difficile strains producing binary toxin, in addition to toxin A (TcdA) and toxin B (TcdB), have been associated with more severe disease and increased recurrence of C. difficile infection in recent outbreaks. Binary toxin comprises two subunits (CDTa and CDTb) and catalyzes the ADP-ribosylation of globular actin (G-actin), which leads to the depolymerization of filamentous actin (F-actin) filaments. A robust assay is highly desirable for detecting the cytotoxic effect of the toxin and the presence of neutralizing antibodies in animal and human sera to evaluate vaccine efficacy. We describe here the optimization, using design-of-experiment (DOE) methodology, of a high-throughput assay to measure the toxin potency and neutralizing antibodies (NAb) against binary toxin. Vero cells were chosen from a panel of cells screened for sensitivity and specificity. We have successfully optimized the CDTa-to-CDTb molar ratio, toxin concentration, cell-seeding density, and sera-toxin preincubation time in the NAb assay using DOE methodology. This assay is robust, produces linear results across serial dilutions of hyperimmune serum, and can be used to quantify neutralizing antibodies in sera from hamsters and monkeys immunized with C. difficile binary toxin-containing vaccines. The assay will be useful for C. difficile diagnosis, for epidemiology studies, and for selecting and optimizing vaccine candidates.
| S-EPMC4018877 | biostudies-literature