scholarly journals The C-Terminal Domain of Clostridioides difficile TcdC Is Exposed on the Bacterial Cell Surface

2020 ◽  
Vol 202 (22) ◽  
Author(s):  
Ana M. Oliveira Paiva ◽  
Leen de Jong ◽  
Annemieke H. Friggen ◽  
Wiep Klaas Smits ◽  
Jeroen Corver
Keyword(s):  
Gene Expression ◽  
Sigma Factor ◽  
Cysteine Residue ◽  
Toxin Gene ◽  
Cytoplasmic Localization ◽  
Content Type ◽  
Clostridioides Difficile ◽  
C Terminus ◽  
Ob Fold ◽  
Toxin Gene Expression

ABSTRACT Clostridioides difficile is an anaerobic Gram-positive bacterium that can produce the large clostridial toxins toxin A and toxin B, encoded within the pathogenicity locus (PaLoc). The PaLoc also encodes the sigma factor TcdR, which positively regulates toxin gene expression, and TcdC, which is a putative negative regulator of toxin expression. TcdC is proposed to be an anti-sigma factor; however, several studies failed to show an association between the tcdC genotype and toxin production. Consequently, the TcdC function is not yet fully understood. Previous studies have characterized TcdC as a membrane-associated protein with the ability to bind G-quadruplex structures. The binding to the DNA secondary structures is mediated through the oligonucleotide/oligosaccharide binding fold (OB-fold) domain present at the C terminus of the protein. This domain was previously also proposed to be responsible for the inhibitory effect on toxin gene expression, implicating a cytoplasmic localization of the OB-fold. In this study, we aimed to obtain topological information on the C terminus of TcdC and demonstrate that the C terminus of TcdC is located extracellularly. In addition, we show that the membrane association of TcdC is dependent on a membrane-proximal cysteine residue and that mutating this residue results in the release of TcdC from the bacterial cell. The extracellular location of TcdC is not compatible with the direct binding of the OB-fold domain to intracellular nucleic acid or protein targets and suggests a mechanism of action that is different from that of the characterized anti-sigma factors. IMPORTANCE The transcription of C. difficile toxins TcdA and TcdB is directed by the sigma factor TcdR. TcdC has been proposed to be an anti-sigma factor. The activity of TcdC has been mapped to its C terminus, and the N terminus serves as the membrane anchor. Acting as an anti-sigma factor requires a cytoplasmic localization of the C terminus of TcdC. Using cysteine accessibility analysis and a HiBiT-based system, we show that the TcdC C terminus is located extracellularly, which is incompatible with its role as anti-sigma factor. Furthermore, mutating a cysteine residue at position 51 resulted in the release of TcdC from the bacteria. The codon-optimized version of the HiBiT (HiBiTopt) extracellular detection system is a valuable tool for topology determination of membrane proteins, increasing the range of systems available to tackle important aspects of C. difficile development.

scholarly journals The C-terminal domain of Clostridioides difficile TcdC is exposed on the bacterial cell surface

2019 ◽  
Author(s):  
Ana M. Oliveira Paiva ◽  
Leen de Jong ◽  
Annemieke H. Friggen ◽  
Wiep Klaas Smits ◽  
Jeroen Corver
Keyword(s):  
Sigma Factor ◽  
Negative Regulator ◽  
Toxin Gene ◽  
Cytoplasmic Localization ◽  
Terminal Domain ◽  
Clostridioides Difficile ◽  
C Terminus ◽  
N Terminus ◽  
Ob Fold ◽  
Toxin Gene Expression

AbstractClostridioides difficile is an anaerobic gram-positive bacterium that can can produce the large clostridial toxins, Toxin A and Toxin B, encoded within the pathogenicity locus (PaLoc). The PaLoc also encodes the sigma factor TcdR, that positively regulates toxin gene expression, and TcdC, a putative negative regulator of toxin expression. TcdC is proposed to be an anti-sigma factor, however, several studies failed to show an association between tcdC genotype and toxin production. Consequently, TcdC function is not yet fully understood. Previous studies have characterized TcdC as a membrane-associated protein with the ability to bind G-quadruplex structures. The binding to the DNA secondary structures is mediated through the OB-fold domain present at the C-terminus of the protein. This domain was previously also proposed to be responsible for the inhibitory effect on toxin gene expression, implicating a cytoplasmic localization of the C-terminal OB-fold.In this study we aimed to obtain topological information on the C-terminus of TcdC. Using Scanning Cysteine Accessibility Mutagenesis and a HiBiT-based system, we demonstrate that the C-terminus of TcdC is located extracellularly. The extracellular location of TcdC is not compatible with direct binding of the OB-fold domain to intracellular nucleic acid or protein targets, and suggests a mechanism of action that is different from characterized anti-sigma factors.ImportanceTranscription of the C. difficile large clostrididial toxins (TcdA and TcdB) is directed by the sigma factor TcdR. TcdC has been implicated as a negative regulator, possible acting as an anti-sigma factor.Activity of TcdC has been mapped to its C-terminal OB fold domain. TcdC is anchored in the bacterial membrane, through its hydrophobic N-terminus and acting as an anti-sigma factor would require cytoplasmic localization of the C-terminal domain.Remarkably, topology predictions for TcdC suggest the N-terminus to be membrane localized and the C-terminal domain to be located extracellularly. Using independent assays, we show that the C-terminus of TcdC indeed is located in the extracellular environment, which is incompatible with its proposed role as anti-sigma factor in toxin regulation.

scholarly journals RstA Is a Major Regulator ofClostridioides difficileToxin Production and Motility

mBio ◽  
2019 ◽  
Vol 10 (2) ◽  
Author(s):  
Adrianne N. Edwards ◽  
Brandon R. Anjuwon-Foster ◽  
Shonna M. McBride
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Toxin Production ◽  
Toxin Gene ◽  
Dna Binding Domain ◽  
Content Type ◽  
Toxin Genes ◽  
Clostridioides Difficile ◽  
Species Specific ◽  
Toxin Gene Expression

ABSTRACTClostridioides difficileinfection (CDI) is a toxin-mediated diarrheal disease. Several factors have been identified that influence the production of the two majorC. difficiletoxins, TcdA and TcdB, but prior published evidence suggested that additional unknown factors were involved in toxin regulation. Previously, we identified aC. difficileregulator, RstA, that promotes sporulation and represses motility and toxin production. We observed that the predicted DNA-binding domain of RstA was required for RstA-dependent repression of toxin genes, motility genes, andrstAtranscription. In this study, we further investigated the regulation of toxin and motility gene expression by RstA. DNA pulldown assays confirmed that RstA directly binds therstApromoter via the predicted DNA-binding domain. Through mutational analysis of therstApromoter, we identified several nucleotides that are important for RstA-dependent transcriptional regulation. Further, we observed that RstA directly binds and regulates the promoters of the toxin genestcdAandtcdB, as well as the promoters for thesigDandtcdRgenes, which encode regulators of toxin gene expression. Complementation analyses with theClostridium perfringensRstA ortholog and a multispecies chimeric RstA protein revealed that theC. difficileC-terminal domain is required for RstA DNA-binding activity, suggesting that species-specific signaling controls RstA function. Our data demonstrate that RstA is a transcriptional repressor that autoregulates its own expression and directly inhibits transcription of the two toxin genes and two positive toxin regulators, thereby acting at multiple regulatory points to control toxin production.IMPORTANCEClostridioides difficileis an anaerobic, gastrointestinal pathogen of humans and other mammals.C. difficileproduces two major toxins, TcdA and TcdB, which cause the symptoms of the disease, and forms dormant endospores to survive the aerobic environment outside the host. A recently discovered regulatory factor, RstA, inhibits toxin production and positively influences spore formation. Herein, we determine that RstA directly binds its own promoter DNA to repress its own gene transcription. In addition, our data demonstrate that RstA directly represses toxin gene expression and gene expression of two toxin gene activators, TcdR and SigD, creating a complex regulatory network to tightly control toxin production. This study provides a novel regulatory link betweenC. difficilesporulation and toxin production. Further, our data suggest thatC. difficiletoxin production is regulated through a direct, species-specific sensing mechanism.

scholarly journals Strain-Dependent RstA Regulation of Clostridioides difficile Toxin Production and Sporulation

2019 ◽  
Vol 202 (2) ◽  
Author(s):  
Adrianne N. Edwards ◽  
Ellen G. Krall ◽  
Shonna M. McBride
Keyword(s):  
Gene Expression ◽  
Gastrointestinal Tract ◽  
Toxin Production ◽  
Spore Formation ◽  
Toxin Gene ◽  
Disease Symptoms ◽  
Content Type ◽  
Clostridioides Difficile ◽  
Toxin Gene Expression ◽  
Strain Dependent

ABSTRACT The anaerobic spore former Clostridioides difficile causes significant diarrheal disease in humans and other mammals. Infection begins with the ingestion of dormant spores, which subsequently germinate within the host gastrointestinal tract. There, the vegetative cells proliferate and secrete two exotoxins, TcdA and TcdB, which cause disease symptoms. Although spore formation and toxin production are critical for C. difficile pathogenesis, the regulatory links between these two physiological processes are not well understood and are strain dependent. Previously, we identified a conserved C. difficile regulator, RstA, that promotes sporulation initiation through an unknown mechanism and directly and indirectly represses toxin and motility gene transcription in the historical isolate 630Δerm. To test whether perceived strain-dependent differences in toxin production and sporulation are mediated by RstA, we created an rstA mutant in the epidemic ribotype 027 strain R20291. RstA affected sporulation and toxin gene expression similarly but more robustly in R20291 than in 630Δerm. In contrast, no effect on motility gene expression was observed in R20291. Reporter assays measuring transcriptional regulation of tcdR, the sigma factor gene essential for toxin gene expression, identified sequence-dependent effects influencing repression by RstA and CodY, a global nutritional sensor, in four diverse C. difficile strains. Finally, sequence- and strain-dependent differences were evident in RstA negative autoregulation of rstA transcription. Altogether, our data suggest that strain-dependent differences in RstA regulation contribute to the sporulation and toxin phenotypes observed in R20291. Our data establish RstA as an important regulator of C. difficile virulence traits. IMPORTANCE Two critical traits of Clostridioides difficile pathogenesis are toxin production, which causes disease symptoms, and spore formation, which permits survival outside the gastrointestinal tract. The multifunctional regulator RstA promotes sporulation and prevents toxin production in the historical strain 630Δerm. Here, we show that RstA exhibits stronger effects on these phenotypes in an epidemic isolate, R20291, and additional strain-specific effects on toxin and rstA expression are evident. Our data demonstrate that sequence-specific differences within the promoter for the toxin regulator TcdR contribute to the regulation of toxin production by RstA and CodY. These sequence differences account for some of the variability in toxin production among isolates and may allow strains to differentially control toxin production in response to a variety of signals.

scholarly journals RstA is a Major Regulator ofClostridioides difficileToxin Production and Motility

2018 ◽  
Author(s):  
Adrianne N. Edwards ◽  
Brandon R. Anjuwon-Foster ◽  
Shonna M. McBride
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Toxin Production ◽  
Binding Domain ◽  
Toxin Gene ◽  
Dna Binding Domain ◽  
Toxin Genes ◽  
Clostridioides Difficile ◽  
Species Specific ◽  
Toxin Gene Expression

ABSTRACTClostridioides difficileinfection (CDI) is a toxin-mediated disease. Several factors have been identified that influence the production of the two majorC. difficiletoxins, TcdA and TcdB, but prior published evidence suggested that additional unknown factors were involved in toxin regulation. Previously, we identified aC. difficileregulator, RstA, that promotes sporulation and represses motility and toxin production. We observed that the predicted DNA-binding domain of RstA was required for RstA-dependent repression of toxin genes, motility genes andrstAtranscription. In this study, we further investigated the regulation of toxin and motility gene expression by RstA. DNA pulldown assays confirmed that RstA directly binds therstApromoter via the predicted DNA-binding domain. Through mutational analysis of therstApromoter, we identified several nucleotides that are important for RstA-dependent transcriptional regulation. Further, we observed that RstA directly binds and regulates the promoters of the toxin genes,tcdAandtcdB, as well as the promoters for thesigDandtcdRgenes, which encode regulators of toxin gene expression. Complementation analyses with theClostridium perfringensRstA ortholog and a multi-species chimeric RstA protein revealed that theC. difficileC-terminal domain is required for RstA DNA-binding activity, suggesting that species-specific signaling controls RstA function. Our data demonstrate that RstA is a transcriptional repressor that autoregulates its own expression and directly inhibits transcription of the two toxin genes and two positive toxin regulators, thereby acting at multiple regulatory points to control toxin production.IMPORTANCEClostridioides difficileis an anaerobic, gastrointestinal pathogen of humans and other mammals.C. difficileproduces two major toxins, TcdA and TcdB, which cause the symptoms of the disease, and forms dormant endospores to survive the aerobic environment outside of the host. A recently discovered regulatory factor, RstA, inhibits toxin production and positively influences spore formation. Herein, we determine that RstA directly represses toxin gene expression and gene expression of two toxin gene activators, TcdR and SigD, creating a complex regulatory network to tightly control toxin production. In addition, the ability for RstA to bind DNA and repress toxin production requires the species-specific domain predicted to respond to small quorum-sensing peptides. This study provides a novel regulatory link betweenC. difficilesporulation and toxin production. Further, our data suggest thatC. difficiletoxin production is regulated through a direct sensing mechanism.

scholarly journals The Alternative Sigma Factor σH Is Required for Toxin Gene Expression by Bacillus anthracis

2006 ◽  
Vol 189 (5) ◽  
pp. 1874-1883 ◽  
Author(s):  
Maria Hadjifrangiskou ◽  
Yahua Chen ◽  
Theresa M. Koehler
Keyword(s):  
Gene Expression ◽  
Bacillus Anthracis ◽  
Sigma Factor ◽  
State Level ◽  
Growth Conditions ◽  
Exponential Phase ◽  
Toxin Gene ◽  
Gene Encoding ◽  
Independent Manner ◽  
Toxin Gene Expression

ABSTRACT Expression of the structural genes for the anthrax toxin proteins is coordinately controlled by host-related signals, such as elevated CO2, and the trans-acting positive regulator AtxA. In addition to these requirements, toxin gene expression is under growth phase regulation. The transition state regulator AbrB represses atxA expression to influence toxin synthesis. During the late exponential phase of growth, when AbrB levels begin to decrease, toxin synthesis increases. Here we report that toxin gene expression also requires the presence of sigH, a gene encoding the RNA polymerase sigma factor associated with development in Bacillus subtilis. In the well-studied B. subtilis system, σH is required for sporulation and other post-exponential-phase processes and is part of a feedback control pathway for abrB expression. Our data indicate that a Bacillus anthracis sigH-null mutant is asporogenous and toxin deficient. Yet the sigma factor is required for toxin gene expression in a manner that is independent of the pathway leading to post-exponential-phase gene expression. σH positively controls atxA in an AbrB-independent manner. These findings, combined with previous observations, suggest that the steady-state level of atxA expression is critical for optimal toxin gene transcription. We propose a model whereby, under toxin-inducing growth conditions, control of toxin gene expression is fine-tuned by the independent effects of σH and AbrB on the expression of atxA.

scholarly journals Control of Clostridium difficile Physiopathology in Response to Cysteine Availability

2016 ◽  
Vol 84 (8) ◽  
pp. 2389-2405 ◽  
Author(s):  
Thomas Dubois ◽  
Marie Dancer-Thibonnier ◽  
Marc Monot ◽  
Audrey Hamiot ◽  
Laurent Bouillaut ◽  
...  
Keyword(s):  
Gene Expression ◽  
Clostridium Difficile ◽  
Molecular Mechanisms ◽  
Sulfur Metabolism ◽  
Toxin Production ◽  
Toxin Gene ◽  
Wild Type ◽  
Control Of Gene Expression ◽  
Content Type ◽  
Toxin Gene Expression

The pathogenicity ofClostridium difficileis linked to its ability to produce two toxins: TcdA and TcdB. The level of toxin synthesis is influenced by environmental signals, such as phosphotransferase system (PTS) sugars, biotin, and amino acids, especially cysteine. To understand the molecular mechanisms of cysteine-dependent repression of toxin production, we reconstructed the sulfur metabolism pathways ofC. difficilestrain 630in silicoand validated some of them by testingC. difficilegrowth in the presence of various sulfur sources. High levels of sulfide and pyruvate were produced in the presence of 10 mM cysteine, indicating that cysteine is actively catabolized by cysteine desulfhydrases. Using a transcriptomic approach, we analyzed cysteine-dependent control of gene expression and showed that cysteine modulates the expression of genes involved in cysteine metabolism, amino acid biosynthesis, fermentation, energy metabolism, iron acquisition, and the stress response. Additionally, a sigma factor (SigL) and global regulators (CcpA, CodY, and Fur) were tested to elucidate their roles in the cysteine-dependent regulation of toxin production. Among these regulators, onlysigLinactivation resulted in the derepression of toxin gene expression in the presence of cysteine. Interestingly, thesigLmutant produced less pyruvate and H2S than the wild-type strain. Unlike cysteine, the addition of 10 mM pyruvate to the medium for a short time during the growth of the wild-type andsigLmutant strains reduced expression of the toxin genes, indicating that cysteine-dependent repression of toxin production is mainly due to the accumulation of cysteine by-products during growth. Finally, we showed that the effect of pyruvate on toxin gene expression is mediated at least in part by the two-component system CD2602-CD2601.

scholarly journals Environmental Response and Autoregulation of Clostridium difficile TxeR, a Sigma Factor for Toxin Gene Expression

2002 ◽  
Vol 184 (21) ◽  
pp. 5971-5978 ◽  
Author(s):  
Nagraj Mani ◽  
Dena Lyras ◽  
Lisa Barroso ◽  
Pauline Howarth ◽  
Tracy Wilkins ◽  
...  
Keyword(s):  
Gene Expression ◽  
Clostridium Difficile ◽  
Growth Phase ◽  
Sigma Factor ◽  
Cellular Growth ◽  
Broth Culture ◽  
Exponential Growth Phase ◽  
Toxin Gene ◽  
Toxin Genes ◽  
Toxin Gene Expression

ABSTRACT TxeR, a sigma factor that directs Clostridium difficile RNA polymerase to recognize the promoters of two major toxin genes, was shown to stimulate its own synthesis. Whether expressed in C. difficile, Clostridium perfringens, or Escherichia coli, TxeR stimulated transcription of fusions of the txeR promoter region to reporter genes. As is the case for the tox genes, txeR expression was responsive to the cellular growth phase and the constituents of the medium. That is, the level of expression in broth culture was low during the exponential growth phase, but rapidly increased as cells approached the stationary phase. In the presence of excess glucose, expression from the txeR promoter was repressed. The results support a model for toxin gene expression in which synthesis of TxeR is induced by specific environmental signals. The increased level of TxeR then permits high-level expression of the toxin genes. The study of txeR gene regulation in C. difficile was made possible by introduction of a mobilizable, replicative plasmid via conjugation with E. coli.

Association between Clostridioides difficile ribotypes, restriction endonuclease analysis types, and toxin gene expression

Anaerobe ◽  
2018 ◽  
Vol 54 ◽  
pp. 140-143
Author(s):  
Hiroki Watanabe ◽  
Yusuke Koizumi ◽  
Asami Matsumoto ◽  
Nobuhiro Asai ◽  
Yuka Yamagishi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Restriction Endonuclease ◽  
Restriction Endonuclease Analysis ◽  
Toxin Gene ◽  
Clostridioides Difficile ◽  
Toxin Gene Expression ◽  
Endonuclease Analysis

scholarly journals RstA Regulation ofClostridioides difficileToxin Production and Sporulation in Phenotypically Diverse Strains

2019 ◽  
Author(s):  
Adrianne N. Edwards ◽  
Ellen G. Krall ◽  
Shonna M. McBride
Keyword(s):  
Gene Expression ◽  
Gastrointestinal Tract ◽  
Diarrheal Disease ◽  
Toxin Production ◽  
Toxin Gene ◽  
Disease Symptoms ◽  
Clostridioides Difficile ◽  
Sequence Dependent ◽  
Toxin Gene Expression ◽  
Strain Dependent

ABSTRACTThe anaerobic spore-former,Clostridioides difficile, causes significant diarrheal disease in humans and other mammals. Infection begins with the ingestion of dormant spores which subsequently germinate within the host gastrointestinal tract. Here, the vegetative cells proliferate and secrete two exotoxins, TcdA and TcdB, which cause disease symptoms. Although spore formation and toxin production are critical forC. difficilepathogenesis, the regulatory links between these two physiological processes are not well understood and are strain-dependent. Previously, we identified a conservedC. difficileregulator, RstA, that promotes sporulation initiation through an unknown mechanism and directly and indirectly represses toxin gene transcription in the historical isolate, 630Δerm. To test whether perceived strain-dependent differences in toxin production and sporulation are mediated by RstA, we created anrstAmutant in the epidemic 027 ribotype strain, R20291. RstA affects sporulation and toxin gene expression similarly in R20291, although more robust regulatory effects are observed in this strain than in 630Δerm. Reporter assays measuring transcriptional regulation oftcdR, the sigma factor essential for toxin gene expression, identified sequence-dependent effects influencing repression by RstA and CodY, a global nutritional sensor, in four diverseC. difficilestrains. We provide evidence that RstA contributes totcdRbistability in R20291 by biasing cells to a toxin-OFF state. Finally, sequence-dependent and strain-dependent differences were evident in RstA negative autoregulation ofrstAtranscription. Our data establish RstA as an important regulator ofC. difficilevirulence traits, and implicate RstA as a contributor to the variety of sporulation and toxin phenotypes observed in distinct isolates.IMPORTANCETwo critical traits ofClostridioides difficilepathogenesis are the production of toxins, which cause disease symptoms, and the formation of spores, which permit survival outside of the gastrointestinal tract. The multifunctional regulator, RstA, promotes sporulation and prevents toxin production in the historical strain, 630Δerm. Here, we show that RstA functions similarly in an epidemic isolate, R20291, although strain-specific effects on toxin andrstAexpression are evident. Our data demonstrate that sequence-specific differences within the promoter for the toxin regulator, TcdR, contribute to regulation of toxin production by RstA and CodY. These sequence differences account for some of the variability in toxin production among isolates, and may allow strains to differentially control toxin production in response to a variety of signals.

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