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
Publications
Structure of the cell-binding component of the <i>Clostridium difficile</i> binary toxin reveals a di-heptamer macromolecular assembly.
Xu Xingjian X Godoy-Ruiz Raquel R Adipietro Kaylin A KA Peralta Christopher C Ben-Hail Danya D Varney Kristen M KM Cook Mary E ME Roth Braden M BM Wilder Paul T PT Cleveland Thomas T Grishaev Alexander A Neu Heather M HM Michel Sarah L J SLJ Yu Wenbo W Beckett Dorothy D Rustandi Richard R RR Lancaster Catherine C Loughney John W JW Kristopeit Adam A Christanti Sianny S Olson Jessica W JW MacKerell Alexander D AD Georges Amedee des AD Pozharski Edwin E Weber David J DJ
Proceedings of the National Academy of Sciences of the United States of America 20200102 2
Targeting <i>Clostridium difficile</i> infection is challenging because treatment options are limited, and high recurrence rates are common. One reason for this is that hypervirulent <i>C. difficile</i> strains often have a binary toxin termed the <i>C. difficile</i> toxin, in addition to the enterotoxins TsdA and TsdB. The <i>C. difficile</i> toxin has an enzymatic component, termed CDTa, and a pore-forming or delivery subunit termed CDTb. CDTb was characterized here using a combination of sing ...[more]
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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.
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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: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: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 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:Virulence of Clostridium difficile is primarily attributed to the large clostridial toxins A and B while the role of binary toxin (CDT) remains unclear. The prevalence of human strains of C. difficile possessing only CDT genes (A-B-CDT+) is generally low (< 5%), however, this genotype is commonly found in neonatal livestock both in Australia and elsewhere. Zoonotic transmission of C. difficile has been suggested previously. Most human diagnostic tests will not detect A-B-CDT+ strains of C. difficile because they focus on detection of toxin A and/or B. We performed a prospective investigation into the prevalence and genetic characteristics of A-B-CDT+ C. difficile in symptomatic humans. All glutamate dehydrogenase or toxin B gene positive faecal specimens from symptomatic inpatients over 30 days (n = 43) were cultured by enrichment, and C. difficile PCR ribotypes (RTs) and toxin gene profiles determined. From 39 culture-positive specimens, 43 C. difficile isolates were recovered, including two A-B-CDT+ isolates. This corresponded to an A-B-CDT+ prevalence of 2/35 (5.7%) isolates possessing at least one toxin, 2/10 (20%) A-B- isolates, 2/3 CDT+ isolates and 1/28 (3.6%) presumed true CDI cases. No link to Australian livestock-associated C. difficile was found. Neither A-B-CDT+ isolate was the predominant A-B-CDT+ strain found in Australia, RT 033, nor did they belong to toxinotype XI. Previous reports infrequently describe A-B-CDT+ C. difficile in patients and strain collections but the prevalence of human A-B-CDT+ C. difficile is rarely investigated. This study highlights the occurrence of A-B-CDT+ strains of C. difficile in symptomatic patients, warranting further investigations of its role in human infection.
| S-EPMC5678700 | biostudies-literature
Project description:A broad spectrum of symptoms has been associated with C. difficile infection (CDI). Several studies indicate that toxin-production correlates with growth rates of C. difficile. This study aimed to correlate growth rates of C. difficile with disease severity and strain characteristics. From 01/2003 to 10/2011, strains from a prospective cohort of all inpatients with CDI at the University Hospital Basel, Switzerland were analyzed regarding binary toxin, presence of the tcdC deletion and ribotype. Isothermal microcalorimetry was performed to determine growth rates, quantified by the Gompertz function. Ordered logistic regression models were used to correlate disease severity with strain features and clinical characteristics. Among 199 patients, 31 (16%) were infected with binary toxin-producing strains, of which the tcdC gene-deletion nt117 was detected in 9 (4%). Disease severity was classified as mild in 130 patients (65.3%), as severe in 59 patients (29.7%) and as severe/complicated in 10 patients (5.0%). Growth rates were inversely associated with disease severity in univariable (OR 0.514, 95%CI 0.29-0.91, p = 0.023) and multivariable analyses (OR 0.51, 95%CI 0.26-0.97, p = 0.040). While none of the strain characteristics such as presence of the tcdC gene deletion or binary toxin predicted CDI severity, growth rates were inversely correlated with disease severity. Further investigations are needed to analyze growth-regulators and respective correlations with the level of toxin production in C. difficile, which may be important determinants of disease severity.
| S-EPMC5012633 | biostudies-literature
Project description:The spore-forming gut bacterium Clostridium difficile is the leading cause of antibiotic-associated diarrhea in hospitalized patients. The major virulence factors are two large glucosylating cytotoxins. Hypervirulent strains (e.g. ribotype 027) with higher morbidity and mortality additionally produce the binary CDT toxin (Clostridium difficile transferase) that ADP-ribosylates actin and induces microtubule-based cell protrusions. Nanobodies are robust single domain antibodies derived from camelid heavy chain antibodies. Here we report the generation of functional nanobodies against the enzymatic CDTa and the heptameric receptor binding subunit CDTb. The nanobodies were obtained from a variable-domain repertoire library isolated from llamas immunized with recombinant CDTa or CDTb. Five CDTa-specific nanobodies blocked CDTa-mediated ADP-ribosylation of actin. Three CDTa-specific and two CDTb-specific nanobodies neutralized the cytotoxicity of CDTa+b. These nanobodies hold promise as new tools for research, diagnosis and therapy of C. difficile associated disease.
| S-EPMC4297958 | biostudies-literature