scholarly journals The Binary Toxin of Clostridioides difficile Alters the Proteome and Phosphoproteome of HEp-2 Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Florian Stieglitz ◽  
Ralf Gerhard ◽  
Andreas Pich
Keyword(s):  
Pseudomembranous Colitis ◽  
Binary Toxin ◽  
Target Cells ◽  
Toxin B ◽  
Clostridioides Difficile ◽  
Antibiotic Associated Diarrhea ◽  
Proteome Level ◽  
Underlying Mechanisms ◽  
Adp Ribosyltransferase ◽  
First Time

Clostridioides difficile is a major cause of nosocomial infection worldwide causing antibiotic-associated diarrhea and some cases are leading to pseudomembranous colitis. The main virulence factors are toxin A and toxin B. Hypervirulent strains of C. difficile are linked to higher mortality rates and most of these strains produce additionally the C. difficile binary toxin (CDT) that possesses two subunits, CDTa and CDTb. The latter is responsible for binding and transfer of CDTa into the cytoplasm of target cells; CDTa is an ADP ribosyltransferase catalyzing the modification of actin fibers that disturbs the actin vs microtubule balance and induces microtubule-based protrusions of the cell membrane increasing the adherence of C. difficile. The underlying mechanisms remain elusive. Thus, we performed a screening experiment using MS-based proteomics and phosphoproteomics techniques. Epithelial Hep-2 cells were treated with CDTa and CDTb in a multiplexed study for 4 and 8 h. Phosphopeptide enrichment was performed using affinity chromatography with TiO2 and Fe-NTA; for quantification, a TMT-based approach and DDA measurements were used. More than 4,300 proteins and 5,600 phosphosites were identified and quantified at all time points. Although only moderate changes were observed on proteome level, the phosphorylation level of nearly 1,100 phosphosites responded to toxin treatment. The data suggested that CSNK2A1 might act as an effector kinase after treatment with CDT. Additionally, we confirmed ADP-ribosylation on Arg-177 of actin and the kinetic of this modification for the first time.

scholarly journals Binary Toxin Production in Clostridium difficile Is Regulated by CdtR, a LytTR Family Response Regulator

2007 ◽  
Vol 189 (20) ◽  
pp. 7290-7301 ◽  
Author(s):  
Glen P. Carter ◽  
Dena Lyras ◽  
David L. Allen ◽  
Kate E. Mackin ◽  
Pauline M. Howarth ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Response Regulator ◽  
Toxin Production ◽  
Regulatory Control ◽  
Binary Toxin ◽  
Response Regulators ◽  
Toxin B ◽  
Adp Ribosyltransferase ◽  
First Time

ABSTRACT 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.

scholarly journals Production of p-cresol by Decarboxylation of p-HPA by All Five Lineages of Clostridioides difficile Provides a Growth Advantage

2021 ◽  
Vol 11 ◽  
Author(s):  
Mark A. Harrison ◽  
Harparkash Kaur ◽  
Brendan W. Wren ◽  
Lisa F. Dawson
Keyword(s):  
Pseudomembranous Colitis ◽  
Bacterial Species ◽  
Host Cells ◽  
Gram Negative ◽  
Clostridioides Difficile ◽  
Antibiotic Associated Diarrhea ◽  
Tyrosine Metabolism ◽  
Hydroxyphenylacetic Acid ◽  
Intracellular Phosphate ◽  
Differential Efficiency

Clostridioides difficile is the leading cause of antibiotic-associated diarrhea and is capable of causing severe symptoms, such as pseudomembranous colitis and toxic megacolon. An unusual feature of C. difficile is the distinctive production of high levels of the antimicrobial compound para-cresol. p-Cresol production provides C. difficile with a competitive colonization advantage over gut commensal species, in particular, Gram-negative species. p-Cresol is produced by the conversion of para-hydroxyphenylacetic acid (p-HPA) via the actions of HpdBCA decarboxylase coded by the hpdBCA operon. Host cells and certain bacterial species produce p-HPA; however, the effects of p-HPA on the viability of C. difficile and other gut microbiota are unknown. Here we show that representative strains from all five C. difficile clades are able to produce p-cresol by two distinct mechanisms: (i) via fermentation of p-tyrosine and (ii) via uptake and turnover of exogenous p-HPA. We observed strain-specific differences in p-cresol production, resulting from differential efficiency of p-tyrosine fermentation; representatives of clade 3 (CD305) and clade 5 (M120) produced the highest levels of p-cresol via tyrosine metabolism, whereas the toxin A-/B+ isolate from clade 4 (M68) produced the lowest level of p-cresol. All five lineages share at least 97.3% homology across the hpdBCA operon, responsible for decarboxylation of p-HPA to p-cresol, suggesting that the limiting step in p-cresol production may result from tyrosine to p-HPA conversion. We identified that elevated intracellular p-HPA, modulated indirectly via CodY, controls p-cresol production via inducing the expression of HpdBCA decarboxylase ubiquitously in C. difficile populations. Efficient turnover of p-HPA is advantageous to C. difficile as p-HPA has a deleterious effect on the growth of C. difficile and other representative Gram-negative gut bacteria, transduced potentially by the disruption of membrane permeability and release of intracellular phosphate. This study provides insights into the importance of HpdBCA decarboxylase in C. difficile pathogenesis, both in terms of p-cresol production and detoxification of p-HPA, highlighting its importance to cell survival and as a highly specific therapeutic target for the inhibition of p-cresol production across C. difficile species.

scholarly journals Functional analyses of epidemic Clostridioides difficile toxin B variants reveal their divergence in utilizing receptors and inducing pathology

2021 ◽  
Vol 17 (1) ◽  
pp. e1009197
Author(s):  
Zhenrui Pan ◽  
Yuanyuan Zhang ◽  
Jianhua Luo ◽  
Danyang Li ◽  
Yao Zhou ◽  
...  
Keyword(s):  
Virulence Factor ◽  
Pseudomembranous Colitis ◽  
Inflammatory Cell ◽  
Protease Domain ◽  
Toxin B ◽  
Clostridioides Difficile ◽  
Sequence Variations ◽  
Cysteine Protease Domain ◽  
Functional Analyses ◽  
Chimeric Toxins

Clostridioides difficile toxin B (TcdB) is a key virulence factor that causes C. difficile associated diseases (CDAD) including diarrhea and pseudomembranous colitis. TcdB can be divided into multiple subtypes/variants based on their sequence variations, of which four (TcdB1-4) are dominant types found in major epidemic isolates. Here, we find that these variants are highly diverse in their receptor preference: TcdB1 uses two known receptors CSPG4 and Frizzled (FZD) proteins, TcdB2 selectively uses CSPG4, TcdB3 prefers to use FZDs, whereas TcdB4 uses neither CSPG4 nor FZDs. By creating chimeric toxins and systematically switching residues between TcdB1 and TcdB3, we determine that regions in the N-terminal cysteine protease domain (CPD) are involved in CSPG4-recognition. We further evaluate the pathological effects induced by TcdB1-4 with a mouse intrarectal installation model. TcdB1 leads to the most severe overall symptoms, followed by TcdB2 and TcdB3. When comparing the TcdB2 and TcdB3, TcdB2 causes stronger oedema while TcdB3 induces severer inflammatory cell infiltration. These findings together demonstrate divergence in the receptor preference and further lead to colonic pathology for predominant TcdB subtypes.

Super toxins from a super bug: structure and function of Clostridium difficile toxins

2011 ◽  
Vol 436 (3) ◽  
pp. 517-526 ◽  
Author(s):  
Abigail H. Davies ◽  
April K. Roberts ◽  
Clifford C. Shone ◽  
K. Ravi Acharya
Keyword(s):  
Cell Death ◽  
Clostridium Difficile ◽  
Rho Gtpases ◽  
Pseudomembranous Colitis ◽  
Regulatory Proteins ◽  
Binary Toxin ◽  
Target Cells ◽  
Transport Component ◽  
Underlying Cause Of Death ◽  
And Function

Clostridium difficile, a highly infectious bacterium, is the leading cause of antibiotic-associated pseudomembranous colitis. In 2009, the number of death certificates mentioning C. difficile infection in the U.K. was estimated at 3933 with 44% of certificates recording infection as the underlying cause of death. A number of virulence factors facilitate its pathogenicity, among which are two potent exotoxins; Toxins A and B. Both are large monoglucosyltransferases that catalyse the glucosylation, and hence inactivation, of Rho-GTPases (small regulatory proteins of the eukaryote actin cell cytoskeleton), leading to disorganization of the cytoskeleton and cell death. The roles of Toxins A and B in the context of C. difficile infection is unknown. In addition to these exotoxins, some strains of C. difficile produce an unrelated ADP-ribosylating binary toxin. This toxin consists of two independently produced components: an enzymatic component (CDTa) and the other, the transport component (CDTb) which facilitates translocation of CDTa into target cells. CDTa irreversibly ADP-ribosylates G-actin in target cells, which disrupts the F-actin:G-actin equilibrium leading to cell rounding and cell death. In the present review we provide a summary of the current structural understanding of these toxins and discuss how it may be used to identify potential targets for specific drug design.

scholarly journals The prognostic value of toxin B and binary toxin in Clostridioides difficile infection

Gut Microbes ◽  
2021 ◽  
pp. 1-8
Author(s):  
Salvador López-Cárdenas ◽  
Eva Torres-Martos ◽  
Juan Mora-Delgado ◽  
Juan Manuel Sánchez-Calvo ◽  
Marta Santos-Peña ◽  
...  
Keyword(s):  
Prognostic Value ◽  
Binary Toxin ◽  
Toxin B ◽  
Clostridioides Difficile ◽  
Clostridioides Difficile Infection

scholarly journals The antimicrobial potential of cannabidiol

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Mark A. T. Blaskovich ◽  
Angela M. Kavanagh ◽  
Alysha G. Elliott ◽  
Bing Zhang ◽  
Soumya Ramu ◽  
...  
Keyword(s):  
Bacterial Infections ◽  
Modern Medicine ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
New Class ◽  
Clostridioides Difficile ◽  
Excellent Activity ◽  
Induce Resistance ◽  
First Time

AbstractAntimicrobial resistance threatens the viability of modern medicine, which is largely dependent on the successful prevention and treatment of bacterial infections. Unfortunately, there are few new therapeutics in the clinical pipeline, particularly for Gram-negative bacteria. We now present a detailed evaluation of the antimicrobial activity of cannabidiol, the main non-psychoactive component of cannabis. We confirm previous reports of Gram-positive activity and expand the breadth of pathogens tested, including highly resistant Staphylococcus aureus, Streptococcus pneumoniae, and Clostridioides difficile. Our results demonstrate that cannabidiol has excellent activity against biofilms, little propensity to induce resistance, and topical in vivo efficacy. Multiple mode-of-action studies point to membrane disruption as cannabidiol’s primary mechanism. More importantly, we now report for the first time that cannabidiol can selectively kill a subset of Gram-negative bacteria that includes the ‘urgent threat’ pathogen Neisseria gonorrhoeae. Structure-activity relationship studies demonstrate the potential to advance cannabidiol analogs as a much-needed new class of antibiotics.

scholarly journals The Importance of Therapeutically Targeting the Binary Toxin from Clostridioides difficile

2021 ◽  
Vol 22 (6) ◽  
pp. 2926
Author(s):  
Dinendra L. Abeyawardhane ◽  
Raquel Godoy-Ruiz ◽  
Kaylin A. Adipietro ◽  
Kristen M. Varney ◽  
Richard R. Rustandi ◽  
...  
Keyword(s):  
Host Cell ◽  
The Elderly ◽  
Cell Receptor ◽  
Host Cells ◽  
Binary Toxin ◽  
Clostridioides Difficile ◽  
Host Cell Receptor ◽  
Oligomeric Assembly ◽  
Nosocomial Disease ◽  
Binary Toxins

Novel therapeutics are needed to treat pathologies associated with the Clostridioides difficile binary toxin (CDT), particularly when C. difficile infection (CDI) occurs in the elderly or in hospitalized patients having illnesses, in addition to CDI, such as cancer. While therapies are available to block toxicities associated with the large clostridial toxins (TcdA and TcdB) in this nosocomial disease, nothing is available yet to treat toxicities arising from strains of CDI having the binary toxin. Like other binary toxins, the active CDTa catalytic subunit of CDT is delivered into host cells together with an oligomeric assembly of CDTb subunits via host cell receptor-mediated endocytosis. Once CDT arrives in the host cell’s cytoplasm, CDTa catalyzes the ADP-ribosylation of G-actin leading to degradation of the cytoskeleton and rapid cell death. Although a detailed molecular mechanism for CDT entry and host cell toxicity is not yet fully established, structural and functional resemblances to other binary toxins are described. Additionally, unique conformational assemblies of individual CDT components are highlighted herein to refine our mechanistic understanding of this deadly toxin as is needed to develop effective new therapeutic strategies for treating some of the most hypervirulent and lethal strains of CDT-containing strains of CDI.

scholarly journals Proteomic Adaptation of Clostridioides difficile to Treatment with the Antimicrobial Peptide Nisin

Cells ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 372
Author(s):  
Sandra Maaß ◽  
Jürgen Bartel ◽  
Pierre-Alexander Mücke ◽  
Rabea Schlüter ◽  
Thomas Sura ◽  
...  
Keyword(s):  
State Of The Art ◽  
Biomedical Application ◽  
Time Resolved ◽  
Antibiotic Resistant ◽  
Cellular Processes ◽  
Clostridioides Difficile ◽  
Antibiotic Associated Diarrhea ◽  
Life Threatening ◽  
Sublethal Concentrations ◽  
Interesting Alternative

Clostridioides difficile is the leading cause of antibiotic-associated diarrhea but can also result in more serious, life-threatening conditions. The incidence of C. difficile infections in hospitals is increasing, both in frequency and severity, and antibiotic-resistant C. difficile strains are advancing. Against this background antimicrobial peptides (AMPs) are an interesting alternative to classic antibiotics. Information on the effects of AMPs on C. difficile will not only enhance the knowledge for possible biomedical application but may also provide insights into mechanisms of C. difficile to adapt or counteract AMPs. This study applies state-of-the-art mass spectrometry methods to quantitatively investigate the proteomic response of C. difficile 630∆erm to sublethal concentrations of the AMP nisin allowing to follow the cellular stress adaptation in a time-resolved manner. The results do not only point at a heavy reorganization of the cellular envelope but also resulted in pronounced changes in central cellular processes such as carbohydrate metabolism. Further, the number of flagella per cell was increased during the adaptation process. The potential involvement of flagella in nisin adaptation was supported by a more resistant phenotype exhibited by a non-motile but hyper-flagellated mutant.

scholarly journals Colonisation Factor CD0873, an Attractive Oral Vaccine Candidate against Clostridioides difficile

2021 ◽  
Vol 9 (2) ◽  
pp. 306
Author(s):  
Cansu Karyal ◽  
Jaime Hughes ◽  
Michelle L. Kelly ◽  
Jeni C. Luckett ◽  
Philip V. Kaye ◽  
...  
Keyword(s):  
Pseudomembranous Colitis ◽  
Vaccine Candidate ◽  
Oral Vaccine ◽  
Immunoglobulin A ◽  
Oral Route ◽  
Hamster Model ◽  
Clostridioides Difficile ◽  
Mucosal Antibody ◽  
Gut Pathogens ◽  
Secretory Immunoglobulin

Clostridioides difficile is the main cause of health-care-associated infectious diarrhoea. Toxins, TcdA and TcdB, secreted by this bacterium damage colonic epithelial cells and in severe cases this culminates in pseudomembranous colitis, toxic megacolon and death. Vaccines in human trials have focused exclusively on the parenteral administration of toxin-based formulations. These vaccines promote toxin-neutralising serum antibodies but fail to confer protection from infection in the gut. An effective route to immunise against gut pathogens and stimulate a protective mucosal antibody response (secretory immunoglobulin A, IgA) at the infection site is the oral route. Additionally, oral immunisation generates systemic antibodies (IgG). Using this route, two different antigens were tested in the hamster model: The colonisation factor CD0873 and a TcdB fragment. Animals immunised with CD0873 generated a significantly higher titre of sIgA in intestinal fluid and IgG in serum compared to naive animals, which significantly inhibited the adherence of C. difficile to Caco-2 cells. Following challenge with a hypervirulent isolate, the CD0873-immunised group showed a mean increase of 80% in time to experimental endpoint compared to naïve animals. Survival and body condition correlated with bacterial clearance and reduced pathology in the cecum. Our findings advocate CD0873 as a promising oral vaccine candidate against C. difficile.

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