scholarly journals Structure of the cell-binding component of the Clostridium difficile binary toxin reveals a novel macromolecular assembly

2019 ◽  
Author(s):  
Xingjian Xu ◽  
Raquel Godoy-Ruiz ◽  
Kaylin A. Adipietro ◽  
Christopher Peralta ◽  
Danya Ben-Hail ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Treatment Options ◽  
The United States ◽  
Atomic Resolution ◽  
Binary Toxin ◽  
Cell Binding ◽  
Recurrence Rates ◽  
Binding Domains ◽  
Starting Point ◽  
Binding Component

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.

scholarly journals Structure of the cell-binding component of the Clostridium difficile binary toxin reveals a di-heptamer macromolecular assembly

2020 ◽  
Vol 117 (2) ◽  
pp. 1049-1058 ◽  
Author(s):  
Xingjian Xu ◽  
Raquel Godoy-Ruiz ◽  
Kaylin A. Adipietro ◽  
Christopher Peralta ◽  
Danya Ben-Hail ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Receptor Binding ◽  
Treatment Options ◽  
Binding Domain ◽  
Binary Toxin ◽  
Receptor Binding Domain ◽  
Design Strategies ◽  
Recurrence Rates ◽  
Structure Based Drug Design ◽  
Starting Point

Targeting Clostridium difficile infection is challenging because treatment options are limited, and high recurrence rates are common. One reason for this is that hypervirulent C. difficile strains often have a binary toxin termed the C. difficile toxin, in addition to the enterotoxins TsdA and TsdB. The C. difficile toxin 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 cryoelectron microscopy, X-ray crystallography, NMR, and other biophysical methods. In the absence of CDTa, 2 di-heptamer structures for activated CDTb (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 2 receptor-binding domains of activated CDTb were of particular interest since the receptor-binding domain 1 lacks sequence homology to any other known toxin, and the receptor-binding domain 2 is completely absent in other well-studied heptameric toxins (i.e., anthrax). For AsymCDTb, a Ca2+ binding site was discovered in the first receptor-binding domain that is important for its stability, and the second receptor-binding domain was found to be critical for host cell toxicity and the di-heptamer fold for both forms of activated CDTb. Together, these studies represent a starting point for developing structure-based drug-design strategies to target the most severe strains of C. difficile.

scholarly journals Zinc Level and Its Role in Recurrent Clostridium difficile Infection: A Case Report and Literature Review

2020 ◽  
Vol 8 ◽  
pp. 232470962094131
Author(s):  
Swetha Parvataneni ◽  
Avinash R. Dasari
Keyword(s):  
United States ◽  
Case Report ◽  
Clostridium Difficile ◽  
Clostridium Difficile Infection ◽  
Zinc Supplementation ◽  
The United States ◽  
Zinc Level ◽  
Recurrence Rates ◽  
Oral Transmission ◽  
Recurrent Clostridium Difficile Infection

Clostridium difficile infection is a common nosocomial infection in US hospitals, accounting for approximately 12 800 deaths annually in the United States. These infections are often associated with the use of antibiotics, which can alter the gut microbiome and thus render patients susceptible to C difficile infection. C difficile is often spread via fecal oral transmission. Multiple medications have been developed, but recurrence rates reach 60% after treatment. Recent data have shown that zinc supplementation decreases the recurrence of C difficile infection. In this article, we present a case of recurrent C difficile infection with zinc deficiency in which zinc supplementation improved the symptoms and reduced the incidence of recurrence.

scholarly journals 669. Twelve-Month Durability of Microbiota-Based Therapy RBX2660 for Prevention of Recurrent Clostridium difficile Infection

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S306-S306
Author(s):  
Courtney Jones ◽  
Sarah Mische ◽  
Ken Blount ◽  
Bill Shannon
Keyword(s):  
Clostridium Difficile ◽  
Treatment Options ◽  
Operational Taxonomic Unit ◽  
The United States ◽  
Historical Control ◽  
Control Group ◽  
Phase 2 ◽  
Open Label ◽  
Two Phase

Abstract Background Recurrent Clostridium difficile infections (rCDI) are a public health threat with insufficient treatment options at present. Two Phase 2 clinical studies have reported the efficacy of RBX2660, a standardized, stabilized microbiota-based drug, in preventing rCDI. For one of these trials, we report herein the durability of clinical response (lack of CDI recurrence) and microbiome restoration to 12 months after RBX2660 treatment. Methods Data were drawn from an interim analysis of a multicenter, open-label Phase 2 study in which participants with multi-recurrent rCDI received up to 2 doses of RBX2660 delivered via enema 7 days apart; this analysis includes data to 12 months after treatment, with follow-up ongoing. Efficacy was defined as the absence of CDI recurrence to 56 days after the last dose; and durability is defined as a continued lack of reported recurrence. Participant stool samples collected prior to and at 1, 7, 30, 60 days and 6 and 12 months after treatment were sequenced using a shallow shotgun method, with only treatment responders reported herein. Operational taxonomic unit (OTU) data were used to calculate relative abundance at the class level and Microbiome Health Indices. Results This study included 149 RBX2660-treated participants and 110 historical control patients, in the United States and Canada. As previously reported, the efficacy of RBX2660 in preventing rCDI (79.9%; 119/149) was higher than CDI-free rates in the historical control group (51.8%, 57/110; P < 0.001). Of 109 participants who had a 6-month follow-up, 97.2% (106/109) remained CDI-free, and no new CDI recurrences were reported at 12 months. Among treatment responders, the microbiome composition was restored after treatment to predominance by Bacteroidia- and Clostridia-class bacteria, and these compositions remained stable to 12 months after treatment among participants who provided samples. Conclusion RBX2660, a microbiota-based drug, was efficacious for preventing rCDI, with clinical and microbiome restoration durability to at least 12 months after treatment. The follow-up of efficacy, safety, and microbiome restoration are ongoing. Disclosures All authors: No reported disclosures.

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

mBio ◽  
2013 ◽  
Vol 4 (3) ◽  
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.

scholarly journals Functional properties of the carboxy-terminal host cell-binding domains of the two toxins, TcdA and TcdB, expressed by Clostridium difficile

Glycobiology ◽  
2008 ◽  
Vol 18 (9) ◽  
pp. 698-706 ◽  
Author(s):  
T. Dingle ◽  
S. Wee ◽  
G. L Mulvey ◽  
A. Greco ◽  
E. N Kitova ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Host Cell ◽  
Functional Properties ◽  
Cell Binding ◽  
Binding Domains ◽  
Carboxy Terminal

scholarly journals U.S.-Based National Sentinel Surveillance Study for the Epidemiology of Clostridium difficile-Associated Diarrheal Isolates and Their Susceptibility to Fidaxomicin

2015 ◽  
Vol 59 (10) ◽  
pp. 6437-6443 ◽  
Author(s):  
D. R. Snydman ◽  
L. A. McDermott ◽  
N. V. Jacobus ◽  
C. Thorpe ◽  
S. Stone ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Antimicrobial Susceptibility ◽  
The United States ◽  
Surveillance Study ◽  
Binary Toxin ◽  
Toxin Gene ◽  
Gene Profile ◽  
Geographic Variations ◽  
Convenience Sample ◽  
Content Type

ABSTRACTIn 2011 a surveillance study for the susceptibility to fidaxomicin and epidemiology ofClostridium difficileisolates in the United States was undertaken in seven geographically dispersed medical centers. This report encompasses baseline surveillance in 2011 and 2012 on 925 isolates. A convenience sample ofC. difficileisolates or toxin positive stools from patients were referred to a central laboratory. Antimicrobial susceptibility was determined by agar dilution (CLSI M11-A8). Clinical and Laboratory Standards Institute (CLSI), Food and Drug Administration, or European Union of Clinical Antimicrobial Susceptibility Testing (EUCAST) breakpoints were applied where applicable. Toxin gene profiles were characterized by multiplex PCR on each isolate. A random sample of 322 strains, stratified by institution, underwent restriction endonuclease analysis (REA). The fidaxomicin MIC90was 0.5 μg/ml for all isolates regardless of REA type or toxin gene profile, and all isolates were inhibited at ≤1.0 μg/ml. By REA typing, BI strains represented 25.5% of the isolates. The toxin gene profile oftcdA,tcdB, andcdtA/Bpositive with atcdC18-bp deletion correlated with BI REA group. Moxifloxacin and clindamycin resistance was increased among either BI or binary toxin-positive isolates. Metronidazole and vancomycin showed reduced susceptibility (EUCAST criteria) in these isolates. Geographic variations in susceptibility, REA group and binary toxin gene presence were observed. Fidaxomicin activity againstC. difficileisolated in a national surveillance study did not change more than 1 year after licensure. This analysis provides baseline results for future comparisons.

Higher tamoxifen use as chemoprophylaxis in patients with ductal carcinoma in situ (DCIS) in the last decade.

2012 ◽  
Vol 30 (27_suppl) ◽  
pp. 191-191
Author(s):  
Erin E. Roesch ◽  
Lai Wei ◽  
Bhuvaneswari Ramaswamy
Keyword(s):  
Hormone Therapy ◽  
Ductal Carcinoma ◽  
Early Stage ◽  
Treatment Options ◽  
Retrospective Chart Review ◽  
Breast Conserving Surgery ◽  
The United States ◽  
Recurrence Rates ◽  
Chi Square ◽  
Significant Difference

191 Background: The incidence of DCIS over the last two decades has increased, with over 62,000 new cases diagnosed annually in the United States. Tailored management of DCIS is still a work in progress and currently treatment options are varied. We sought to identify changes in patterns of care in individual and physician choices for surgery and chemoprophylaxis over the last two decades at our institution. Methods: We performed a retrospective chart review and identified 773 eligible patients using the James Cancer Registry and the NCCN database at The Ohio State University between 1990 and 2010. We compared the proportion of patients undergoing mastectomy vs breast conserving surgery (BCT), use of radiation and hormone therapy, and recurrence rates between the years 1990-2000 and 2001-2010 using Chi-square test. Results: There was no significant difference in race (p=0.21) or age (p=0.09) among patients diagnosed with DCIS between 1990-2000 (Group A, N=462) and 2001-2010 (Group B, N=311). There was no significant difference in mastectomy rates between the two groups (31% vs. 27%, p= 0.26). Patients less than 50 years old were more likely to undergo mastectomy in both groups (p=0.02). Use of radiation therapy following BCT was similar between the two groups (52% vs. 53%, p=0.76). Interestingly more patients received hormone therapy during 2001-2010 than 1990-2000 (48% vs. 26%, p < 0.0001). Patients undergoing mastectomy were less likely to receive radiation (2% vs. 74%, p<0.0001) and hormone therapy (25% vs. 39%, p<0.0001). There was no significant correlation between race and type of surgery. We are in the process of comparing recurrence rates between the two groups which will be reported. Conclusions: Previously published studies have reported higher rates of mastectomy among patients with early stage breast cancer in the recent years. Data from our institution over the last two decades did not corroborate this finding, although interestingly we have found increasing use of tamoxifen therapy for DCIS in the second decade. Understanding current practices is helpful in designing future studies for management of DCIS using novel prognostic assays such as Oncotype Dx assay.

scholarly journals Strategies to Prevent Clostridium difficile Infections in Acute Care Hospitals

2008 ◽  
Vol 29 (S1) ◽  
pp. S81-S92 ◽  
Author(s):  
Erik R. Dubberke ◽  
Dale N. Gerding ◽  
David Classen ◽  
Kathleen M. Arias ◽  
Kelly Podgorny ◽  
...  
Keyword(s):  
United States ◽  
Clostridium Difficile ◽  
Acute Care ◽  
Acute Care Hospitals ◽  
The United States ◽  
Binary Toxin ◽  
Healthcare Associated Infections ◽  
Executive Summary ◽  
Healthcare Associated

Previously published guidelines are available that provide comprehensive recommendations for detecting and preventing healthcare-associated infections. The intent of this document is to highlight practical recommendations in a concise format designed to assist acute care hospitals in implementing and prioritizing their Clostridium difficile infection (CDI) prevention efforts. Refer to the Society for Healthcare Epidemiology of America/Infectious Diseases Society of America “Compendium of Strategies to Prevent Healthcare-Associated Infections” Executive Summary and Introduction and accompanying editorial for additional discussion.1. Increasing rates of CDIC. difficile now rivals methicillin-resistant Staphylococcus aureus (MRSA) as the most common organism to cause healthcare-associated infections in the United States.a. In the United States, the proportion of hospital discharges in which the patient received the International Classification of Diseases, Ninth Revision discharge diagnosis code for CDI more than doubled between 2000 and 2003, and CDI rates continued to increase in 2004 and 2005 (L. C. McDonald, MD, personal communication, July 2007). These increases have been seen in pediatric and adult populations, but elderly individuals have been disproportionately affected. CDI incidence has also increased in Canada and Europe.b. There have been numerous reports of an increase in CDI severity.c. Most reports of increases in the incidence and severity of CDI have been associated with the BI/NAP1/027 strain of C. difficile. This strain produces more toxins A and B in vitro than do many other strains of C. difficile, produces a third toxin (binary toxin), and is highly resistant to fluoroquinolones.

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