Correction for Xu et al., Structure of the cell-binding component of the Clostridium difficile binary toxin reveals a di-heptamer macromolecular assembly

AbstractTargeting Clostridium difficile infection (CDI) is challenging because treatment options are limited, and high recurrence rates are common. One reason for this is that hypervirulent CDI often has a binary toxin termed the C. difficile toxin (CDT), in addition to the enterotoxins TsdA and TsdB. CDT has an enzymatic component, termed CDTa, and a pore-forming or delivery subunit termed CDTb. CDTb was characterized here using a combination of single particle cryoEM, X-ray crystallography, NMR, and other biophysical methods. In the absence of CDTa, two novel di-heptamer structures for activated CDTb (aCDTb; 1.0 MDa) were solved at atomic resolution including a symmetric (SymCDTb; 3.14 Å) and an asymmetric form (AsymCDTb; 2.84 Å). Roles played by two receptor-binding domains of aCDTb were of particular interest since RBD1 lacks sequence homology to any other known toxin, and the RBD2 domain is completely absent in other well-studied heptameric toxins (i.e. anthrax). For AsymCDTb, a novel Ca2+ binding site was discovered in RBD1 that is important for its stability, and RBD2 was found to be critical for host cell toxicity and the novel di-heptamer fold for both forms of aCDTb. Together, these studies represent a starting point for structure-based drug-discovery strategies to targeting CDT in the most severe strains of CDI.SIGNIFICANCE STATEMENTThere is a high burden from C. difficile infection (CDI) throughout the world, and the Center for Disease Control (CDC) reports more than 500,000 cases annually in the United States, resulting in an estimated 15,000 deaths. In addition to the large clostridial toxins, TcdA/TcdB, a third C. difficile binary toxin (CDT) is associated with the most serious outbreaks of drug resistant CDI in the 21st century. Here, structural biology and biophysical approaches were used to characterize the cell binding component of CDT, termed CDTb, at atomic resolution. Surprisingly, two novel structures were solved from a single sample that help to explain the molecular underpinnings of C. difficile toxicity. These structures will also be important for targeting this human pathogen via structure-based therapeutic design methods.

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Clostridium difficile Binary Toxin CDT Induces Clustering of the Lipolysis-Stimulated Lipoprotein Receptor into Lipid Rafts

mBio ◽

10.1128/mbio.00244-13 ◽

2013 ◽

Vol 4 (3) ◽

Cited By ~ 29

Author(s):

Panagiotis Papatheodorou ◽

Daniel Hornuss ◽

Thilo Nölke ◽

Sarah Hemmasi ◽

Jan Castonguay ◽

...

Keyword(s):

Plasma Membrane ◽

Clostridium Difficile ◽

Lipid Rafts ◽

Receptor Binding ◽

Binding Domain ◽

Binary Toxin ◽

Lipoprotein Receptor ◽

Receptor Binding Domain ◽

Content Type ◽

Binding Component

ABSTRACT Clostridium difficile is the leading cause of antibiotics-associated diarrhea and pseudomembranous colitis. Hypervirulent C. difficile strains produce the binary actin-ADP-ribosylating toxin CDT (C. difficile transferase), in addition to the Rho-glucosylating toxins A and B. We recently identified the lipolysis-stimulated lipoprotein receptor (LSR) as the host receptor that mediates uptake of CDT into target cells. Here we investigated in H1-HeLa cells, which ectopically express LSR, the influence of CDT on the plasma membrane distribution of the receptor. We found by fluorescence microscopy that the binding component of CDT (CDTb) induces clustering of LSR into subcompartments of the plasma membrane. Detergent extraction of cells treated with CDTb, followed by sucrose gradient fractionation, uncovered accumulation of LSR in detergent-resistant membranes (DRMs) that contained typical marker proteins of lipid rafts. Membrane cholesterol depletion with methyl-β-cyclodextrin inhibited the association of LSR with DRMs upon addition of CDTb. The receptor-binding domain of CDTb also triggered LSR clustering into DRMs. CDTb-triggered clustering of LSR into DRMs could be confirmed in Caco-2 cells. Our data suggest that CDT forces its receptor to cluster into lipid rafts and that oligomerization of the B component might enhance but is not essential for this process. IMPORTANCE C. difficile binary toxin CDT is a member of the iota-like, actin ADP-ribosylating toxin family. The mechanism that mediates endocytic uptake of these toxins still remains elusive. Previous studies highlighted the importance of lipid rafts for oligomerization of the binding component of these toxins and for cell entry. Recently, the host cell receptor for this toxin family, namely, the lipolysis-stimulated lipoprotein receptor (LSR), has been identified. Our study now demonstrates that the binding component of CDT (CDTb) induces clustering of LSR into lipid rafts. Importantly, LSR clustering is efficiently induced also by the receptor-binding domain of CDTb, suggesting that oligomerization of the B component of CDT is not the main trigger of this process. The current work extends our knowledge on the cooperative play between iota-like toxins and their receptor.

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Characterizations of Clinical Isolates of Clostridium difficile by Toxin Genotypes and by Susceptibility to 12 Antimicrobial Agents, Including Fidaxomicin (OPT-80) and Rifaximin: a Multicenter Study in Taiwan

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00191-12 ◽

2012 ◽

Vol 56 (7) ◽

pp. 3943-3949 ◽

Cited By ~ 34

Author(s):

Chun-Hsing Liao ◽

Wen-Chien Ko ◽

Jang-Jih Lu ◽

Po-Ren Hsueh

Keyword(s):

Clostridium Difficile ◽

Care Setting ◽

Antimicrobial Agents ◽

Teaching Hospitals ◽

Binary Toxin ◽

Content Type ◽

Vancomycin Mics ◽

Major Teaching ◽

Southern Taiwan

ABSTRACTA total of 403 nonduplicate isolates ofClostridium difficilewere collected at three major teaching hospitals representing northern, central, and southern Taiwan from January 2005 to December 2010. Of these 403 isolates, 170 (42.2%) were presumed to be nontoxigenic due to the absence of genes for toxins A or B or binary toxin. The remaining 233 (57.8%) isolates carried toxin A and B genes, and 39 (16.7%) of these also had binary toxin genes. The MIC90of all isolates for fidaxomicin and rifaximin was 0.5 μg/ml (range, ≤0.015 to 0.5 μg/ml) and >128 μg/ml (range, ≤0.015 to >128 μg/ml), respectively. All isolates were susceptible to metronidazole (MIC90of 0.5 μg/ml; range, ≤0.03 to 4 μg/ml). Two isolates had reduced susceptibility to vancomycin (MICs, 4 μg/ml). Only 13.6% of isolates were susceptible to clindamycin (MIC of ≤2 μg/ml). Nonsusceptibility to moxifloxacin (n= 81, 20.1%) was accompanied by single or multiple mutations ingyrAandgyrBgenes in all but eight moxifloxacin-nonsusceptible isolates. Two previously unreportedgyrBmutations might independently confer resistance (MIC, 16 μg/ml), Ser416 to Ala and Glu466 to Lys. Moxifloxacin-resistant isolates were cross-resistant to ciprofloxacin and levofloxacin, but some moxifloxacin-nonsusceptible isolates remained susceptible to gemifloxacin or nemonoxacin at 0.5 μg/ml. This study found the diversity of toxigenic and nontoxigenic strains ofC. difficilein the health care setting in Taiwan. All isolates tested were susceptible to metronidazole and vancomycin. Fidaxomicin exhibited potentin vitroactivity against all isolates tested, while the more than 10% of Taiwanese isolates with rifaximin MICs of ≥128 μg/ml raises concerns.

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Molecular characterization and antimicrobial susceptibility patterns of Clostridium difficile strains isolated from hospitals in south-east Scotland

Journal of Medical Microbiology ◽

10.1099/jmm.0.47176-0 ◽

2007 ◽

Vol 56 (7) ◽

pp. 921-929 ◽

Cited By ~ 40

Author(s):

Esvet Mutlu ◽

Allison J. Wroe ◽

Karla Sanchez-Hurtado ◽

Jon S. Brazier ◽

Ian R. Poxton

Keyword(s):

Antibiotic Resistance ◽

Clostridium Difficile ◽

Restriction Analysis ◽

Common Type ◽

Resistance Pattern ◽

Binary Toxin ◽

Resistance Patterns ◽

The Common ◽

Antibiotic Resistance Patterns ◽

Susceptibility Patterns

Clostridium difficile isolates (n=149) collected in south-east Scotland between August and October 2005 were typed by four different methods and their susceptibility to seven different antibiotics was determined. The aims were to define the types of strain occurring in this region and to determine whether there were any clonal relationships among them with respect to genotype and antibiotic resistance pattern. Ribotyping revealed that 001 was the most common type (n=113, 75.8 %), followed by ribotype 106 (12 isolates, 8.1 %). The majority of the isolates (96.6 %, n=144) were of toxinotype 0, with two toxinotype V isolates and single isolates of toxinotypes I, IV and XIII. PCR and restriction analysis of the fliC gene from 147 isolates gave two restriction patterns: 145 of pattern VII and two of pattern I. Binary toxin genes were detected in only three isolates: two isolates of ribotype 126, toxinotype V, and one isolate of ribotype 023, toxinotype IV. S-types showed more variation, with 64.5 % (n=40) of the common S-type (4939) and 21 % (n=13) of S-type 4741, with six other S-types (one to three isolates each). All ribotype 001 isolates were of the same S-type (4939), with three isolates of other ribotypes being this S-type. No resistance was found to metronidazole or vancomycin, with resistance to tetracycline only found in 4.3 % of the isolates. A high proportion of isolates were resistant to clindamycin (62.9 %), moxifloxacin, ceftriaxone (both 87.1 %) and erythromycin (94.8 %). Resistance to three antibiotics (erythromycin, clindamycin and ceftriaxone) was seen in 66 isolates, with erythromycin, ceftriaxone and moxifloxacin resistance seen in 96 isolates. Resistance to all four of these antibiotics was found in 62 isolates and resistance to five (the above plus tetracycline) in one isolate: a ribotype 001, toxinotype 0 strain. Whilst ribotype 001 was the most commonly encountered type, there was no evidence of clonal relationships when all other typing and antibiotic resistance patterns were taken into account.

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Variant forms of the binary toxin CDT locus and tcdC gene in Clostridium difficile strains

Journal of Medical Microbiology ◽

10.1099/jmm.0.46931-0 ◽

2007 ◽

Vol 56 (3) ◽

pp. 329-335 ◽

Cited By ~ 32

Author(s):

Barbara Geric Stare ◽

Michel Delmée ◽

Maja Rupnik

Keyword(s):

Clostridium Difficile ◽

Binary Toxin

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Evaluation of the Cepheid Xpert C. difficile/Epiand Meridian Bioscienceillumigene C. difficile Assays for Detecting Clostridium difficile Ribotype 033 Strains

Journal of Clinical Microbiology ◽

10.1128/jcm.03297-14 ◽

2014 ◽

Vol 53 (3) ◽

pp. 973-975 ◽

Cited By ~ 14

Author(s):

Grace O. Androga ◽

Alan M. McGovern ◽

Briony Elliott ◽

Barbara J. Chang ◽

Timothy T. Perkins ◽

...

Keyword(s):

Clostridium Difficile ◽

Binary Toxin ◽

Content Type ◽

Toxin Genes ◽

Production Animals

Clostridium difficilePCR ribotype 033 (RT033) is found in the gastrointestinal tracts of production animals and, occasionally, humans. TheillumigeneC. difficileassay (Meridian Bioscience, Inc.) failed to detect any of 52C. difficileRT033 isolates, while all strains signaled positive for the binary toxin genes but were reported as negative forC. difficileby the XpertC. difficile/Epiassay (Cepheid).

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Infection with Toxin A-Negative, Toxin B-Negative, Binary Toxin-Positive Clostridium difficile in a Young Patient with Ulcerative Colitis

Journal of Clinical Microbiology ◽

10.1128/jcm.01810-15 ◽

2015 ◽

Vol 53 (11) ◽

pp. 3702-3704 ◽

Cited By ~ 25

Author(s):

Grace O. Androga ◽

Julie Hart ◽

Niki F. Foster ◽

Adrian Charles ◽

David Forbes ◽

...

Keyword(s):

Ulcerative Colitis ◽

Clostridium Difficile ◽

Young Patient ◽

Diagnostic Methods ◽

Binary Toxin ◽

Toxin A ◽

Toxin B ◽

Content Type ◽

Severe Diarrhea ◽

Clinically Significant

Large clostridial toxin-negative, binary toxin-positive (A−B−CDT+) strains ofClostridium difficileare almost never associated with clinically significantC. difficileinfection (CDI), possibly because such strains are not detected by most diagnostic methods. We report the isolation of an A−B−CDT+ribotype 033 (RT033) strain ofC. difficilefrom a young patient with ulcerative colitis and severe diarrhea.

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Binary toxin and its clinical importance in Clostridium difficile infection, Belgium

European Journal of Clinical Microbiology & Infectious Diseases ◽

10.1007/s10096-016-2719-4 ◽

2016 ◽

Vol 35 (11) ◽

pp. 1741-1747 ◽

Cited By ~ 5

Author(s):

T. Pilate ◽

J. Verhaegen ◽

M. Van Ranst ◽

V. Saegeman

Keyword(s):

Clostridium Difficile ◽

Clostridium Difficile Infection ◽

Clinical Importance ◽

Binary Toxin

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Binding Component of Clostridium perfringens Iota-Toxin Induces Endocytosis in Vero Cells

Infection and Immunity ◽

10.1128/iai.70.4.1909-1914.2002 ◽

2002 ◽

Vol 70 (4) ◽

pp. 1909-1914 ◽

Cited By ~ 47

Author(s):

Masahiro Nagahama ◽

Koichi Nagayasu ◽

Keiko Kobayashi ◽

Jun Sakurai

Keyword(s):

Clostridium Perfringens ◽

Vero Cells ◽

Binary Toxin ◽

Wild Type ◽

Oligomer Formation ◽

Clostridium Perfringens Iota Toxin ◽

Binding Component ◽

Pronase Treatment

ABSTRACT Clostridium perfringens iota-toxin is a binary toxin consisting of two individual proteins, the binding component (Ib) and the enzyme component (Ia). Wild-type Ib bound to Vero cells at 4 and 37°C and formed oligomers at 37°C but not at 4°C. The Ib-induced K+ release from the cells was dependent on the oligomer formation of Ib in the cells, but the oligomer formation did not induce rounding activity or cytotoxicity. After incubation of the cells with recombinant Ib (rIb) at 37°C, the Ib oligomer in the cell became resistant to pronase treatment with time, but the Ib monomer was sensitive to the treatment. Furthermore, treatment of Vero cells with rIb in the presence of bafilomycin, methylamine, or ethylamine resulted in accumulation of the oligomer in the cells but had no effect on K+ release. Moreover, incubation with Ib plus Ia in the presence of these agents caused no rounding in the cells. These observations suggest that Ib binds to Vero cells, itself oligomerizing to form ion-permeable channels and that the formation of oligomer then induces endocytosis.

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