scholarly journals Effect of tcdR Mutation on Sporulation in the Epidemic Clostridium difficile Strain R20291

mSphere ◽  
2017 ◽  
Vol 2 (1) ◽  
Author(s):  
Brintha P. Girinathan ◽  
Marc Monot ◽  
Daniel Boyle ◽  
Kathleen N. McAllister ◽  
Joseph A. Sorg ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Toxin Production ◽  
Hospitalized Patients ◽  
Host Tissue ◽  
Hospital Environment ◽  
Pivotal Role ◽  
Toxin Gene ◽  
Bacterial Cells ◽  
Significant Morbidity ◽  
Content Type

ABSTRACT C. difficile infects thousands of hospitalized patients every year, causing significant morbidity and mortality. C. difficile spores play a pivotal role in the transmission of the pathogen in the hospital environment. During infection, the spores germinate, and the vegetative bacterial cells produce toxins that damage host tissue. Thus, sporulation and toxin production are two important traits of C. difficile. In this study, we showed that a mutation in tcdR, the toxin gene regulator, affects both toxin production and sporulation in epidemic-type C. difficile strain R20291. Clostridium difficile is an important nosocomial pathogen and the leading cause of hospital-acquired diarrhea. Antibiotic use is the primary risk factor for the development of C. difficile-associated disease because it disrupts normally protective gut flora and enables C. difficile to colonize the colon. C. difficile damages host tissue by secreting toxins and disseminates by forming spores. The toxin-encoding genes, tcdA and tcdB, are part of a pathogenicity locus, which also includes the tcdR gene that codes for TcdR, an alternate sigma factor that initiates transcription of tcdA and tcdB genes. We created a tcdR mutant in epidemic-type C. difficile strain R20291 in an attempt to identify the global role of tcdR. A site-directed mutation in tcdR affected both toxin production and sporulation in C. difficile R20291. Spores of the tcdR mutant were more heat sensitive than the wild type (WT). Nearly 3-fold more taurocholate was needed to germinate spores from the tcdR mutant than to germinate the spores prepared from the WT strain. Transmission electron microscopic analysis of the spores also revealed a weakly assembled exosporium on the tcdR mutant spores. Accordingly, comparative transcriptome analysis showed many differentially expressed sporulation genes in the tcdR mutant compared to the WT strain. These data suggest that regulatory networks of toxin production and sporulation in C. difficile strain R20291 are linked with each other. IMPORTANCE C. difficile infects thousands of hospitalized patients every year, causing significant morbidity and mortality. C. difficile spores play a pivotal role in the transmission of the pathogen in the hospital environment. During infection, the spores germinate, and the vegetative bacterial cells produce toxins that damage host tissue. Thus, sporulation and toxin production are two important traits of C. difficile. In this study, we showed that a mutation in tcdR, the toxin gene regulator, affects both toxin production and sporulation in epidemic-type C. difficile strain R20291.

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 Spo0A Suppresses sin Locus Expression in Clostridioides difficile

mSphere ◽  
2020 ◽  
Vol 5 (6) ◽  
Author(s):  
Babita Adhikari Dhungel ◽  
Revathi Govind
Keyword(s):  
Diarrheal Disease ◽  
Toxin Production ◽  
The United States ◽  
Upstream Region ◽  
Pcr Analysis ◽  
Bacterial Cells ◽  
Master Regulator ◽  
Content Type ◽  
Clostridioides Difficile

ABSTRACT Clostridioides difficile is the leading cause of nosocomial infection and is the causative agent of antibiotic-associated diarrhea. The severity of the disease is directly associated with toxin production, and spores are responsible for the transmission and persistence of the organism. Previously, we characterized sin locus regulators SinR and SinR′ (we renamed it SinI), where SinR is the regulator of toxin production and sporulation. The SinI regulator acts as its antagonist. In Bacillus subtilis, Spo0A, the master regulator of sporulation, controls SinR by regulating the expression of its antagonist, sinI. However, the role of Spo0A in the expression of sinR and sinI in C. difficile had not yet been reported. In this study, we tested spo0A mutants in three different C. difficile strains, R20291, UK1, and JIR8094, to understand the role of Spo0A in sin locus expression. Western blot analysis revealed that spo0A mutants had increased SinR levels. Quantitative reverse transcription-PCR (qRT-PCR) analysis of its expression further supported these data. By carrying out genetic and biochemical assays, we show that Spo0A can bind to the upstream region of this locus to regulates its expression. This study provides vital information that Spo0A regulates the sin locus, which controls critical pathogenic traits such as sporulation, toxin production, and motility in C. difficile. IMPORTANCE Clostridioides difficile is the leading cause of antibiotic-associated diarrheal disease in the United States. During infection, C. difficile spores germinate, and the vegetative bacterial cells produce toxins that damage host tissue. In C. difficile, the sin locus is known to regulate both sporulation and toxin production. In this study, we show that Spo0A, the master regulator of sporulation, controls sin locus expression. Results from our study suggest that Spo0A directly regulates the expression of this locus by binding to its upstream DNA region. This observation adds new detail to the gene regulatory network that connects sporulation and toxin production in this pathogen.

scholarly journals Cyclic Diguanylate Regulates Virulence Factor Genes via Multiple Riboswitches inClostridium difficile

mSphere ◽  
2018 ◽  
Vol 3 (5) ◽  
Author(s):  
Robert W. McKee ◽  
Carissa K. Harvest ◽  
Rita Tamayo
Keyword(s):  
Gene Expression ◽  
Clostridium Difficile ◽  
Biofilm Formation ◽  
Toxin Production ◽  
Intestinal Colonization ◽  
Cyclic Diguanylate ◽  
Signaling Molecule ◽  
Content Type ◽  
Surface Motility

ABSTRACTThe intracellular signaling molecule cyclic diguanylate (c-di-GMP) regulates many processes in bacteria, with a central role in controlling the switch between motile and nonmotile lifestyles. Recent work has shown that inClostridium difficile(also calledClostridioides difficile), c-di-GMP regulates swimming and surface motility, biofilm formation, toxin production, and intestinal colonization. In this study, we determined the transcriptional regulon of c-di-GMP inC. difficile,employing overexpression of a diguanylate cyclase gene to artificially manipulate intracellular c-di-GMP. Consistent with prior work, c-di-GMP regulated the expression of genes involved in swimming and surface motility. c-di-GMP also affected the expression of multiple genes encoding cell envelope proteins, several of which affected biofilm formationin vitro. A substantial proportion of the c-di-GMP regulon appears to be controlled either directly or indirectly via riboswitches. We confirmed the functionality of 11 c-di-GMP riboswitches, demonstrating their effects on downstream gene expression independent of the upstream promoters. The class I riboswitches uniformly functioned as “off” switches in response to c-di-GMP, while class II riboswitches acted as “on” switches. Transcriptional analyses of genes 3′ of c-di-GMP riboswitches over a broad range of c-di-GMP levels showed that relatively modest changes in c-di-GMP levels are capable of altering gene transcription, with concomitant effects on microbial behavior. This work expands the known c-di-GMP signaling network inC. difficileand emphasizes the role of the riboswitches in controlling known and putative virulence factors inC. difficile.IMPORTANCEInClostridium difficile, the signaling molecule c-di-GMP regulates multiple processes affecting its ability to cause disease, including swimming and surface motility, biofilm formation, toxin production, and intestinal colonization. In this study, we used RNA-seq to define the transcriptional regulon of c-di-GMP inC. difficile. Many new targets of c-di-GMP regulation were identified, including multiple putative colonization factors. Transcriptional analyses revealed a prominent role for riboswitches in c-di-GMP signaling. Only a subset of the 16 previously predicted c-di-GMP riboswitches were functionalin vivoand displayed potential variability in their response kinetics to c-di-GMP. This work underscores the importance of studying c-di-GMP riboswitches in a relevant biological context and highlights the role of the riboswitches in controlling gene expression inC. difficile.

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 Environmental Contamination in Households of Patients with Recurrent Clostridium difficile Infection

2016 ◽  
Vol 82 (9) ◽  
pp. 2686-2692 ◽  
Author(s):  
Megan K. Shaughnessy ◽  
Aleh Bobr ◽  
Michael A. Kuskowski ◽  
Brian D. Johnston ◽  
Michael J. Sadowsky ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Fecal Microbiota Transplantation ◽  
Fecal Microbiota ◽  
Hospital Environment ◽  
Molecular Characteristics ◽  
Species Identity ◽  
Fecal Samples ◽  
Content Type ◽  
Environmental Surfaces ◽  
Household Environment

ABSTRACTRecurrentClostridium difficileinfection (R-CDI) is common and difficult to treat, potentially necessitating fecal microbiota transplantation (FMT). AlthoughC. difficilespores persist in the hospital environment and cause infection, little is known about their potential presence or importance in the household environment. Households of R-CDI subjects in the peri-FMT period and of geographically matched and age-matched controls were analyzed for the presence ofC. difficile. Household environmental surfaces and fecal samples from humans and pets in the household were examined. Households of post-FMT subjects were also examined (environmental surfaces only). Participants were surveyed regarding their personal history and household cleaning habits. Species identity and molecular characteristics of presumptiveC. difficileisolates from environmental and fecal samples were determined by using the Pro kit (Remel, USA), Gram staining, PCR, toxinotyping,tcdCgene sequencing, and pulsed-field gel electrophoresis (PFGE). Environmental cultures detectedC. difficileon ≥1 surface in 8/8 (100%) peri-FMT households, versus 3/8 (38%) post-FMT households and 3/8 (38%) control households (P= 0.025). The most commonC. difficile-positive sites were the vacuum (11/27; 41%), toilet (8/30; 27%), and bathroom sink (5/29; 17%).C. difficilewas detected in 3/36 (8%) fecal samples (two R-CDI subjects and one household member). Nine (90%) of 10 households with multipleC. difficile-positive samples had a single genotype present each. In conclusion,C. difficilewas found in the household environment of R-CDI patients, but whether it was found as a cause or consequence of R-CDI is unknown. If household contamination leads to R-CDI, effective decontamination may be protective.

scholarly journals Carriage of Clostridium difficile by Wild Urban Norway Rats (Rattus norvegicus) and Black Rats (Rattus rattus)

2013 ◽  
Vol 80 (4) ◽  
pp. 1299-1305 ◽  
Author(s):  
Chelsea G. Himsworth ◽  
David M. Patrick ◽  
Sunny Mak ◽  
Claire M. Jardine ◽  
Patrick Tang ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Mixed Models ◽  
Generalized Linear Mixed Models ◽  
Rattus Rattus ◽  
Gene Identification ◽  
Toxin Gene ◽  
Content Type ◽  
Domestic Species ◽  
Enteric Infections ◽  
Norway Rats

ABSTRACTClostridium difficileis an important cause of enteric infections in humans. Recently, concerns have been raised regarding whether animals could be a source ofC. difficilespores. Although colonization has been identified in a number of domestic species, the ability of commensal pests to serve as a reservoir forC. difficilehas not been well investigated. The objective of this study was to determine whether urban rats (Rattusspp.) from Vancouver, Canada, carryC. difficile.Clostridium difficilewas isolated from the colon contents of trapped rats and was characterized using ribotyping, toxinotyping, and toxin gene identification. Generalized linear mixed models and spatial analysis were used to characterize the ecology ofC. difficilein rats.Clostridium difficilewas isolated from 95 of 724 (13.1%) rats, although prevalence differed from 0% to 46.7% among city blocks. The odds of beingC. difficilepositive decreased with increasing weight (odds ratio [OR], 0.67; 95% confidence interval [CI], 0.53 to 0.87), suggesting that carriage is more common in younger animals. The strains isolated included 9 ribotypes that matched recognized international designations, 5 identified by our laboratory in previous studies, and 21 “novel” ribotypes. Some strains were clustered geographically; however, the majority were dispersed throughout the study area, supporting environmental sources of exposure and widespread environmental contamination with a variety ofC. difficilestrains. Given that urban rats are the source of a number of other pathogens responsible for human morbidity and mortality, the potential for rats to be a source ofC. difficilefor humans deserves further consideration.

scholarly journals A Nutrient-Regulated Cyclic Diguanylate Phosphodiesterase Controls Clostridium difficile Biofilm and Toxin Production during Stationary Phase

2017 ◽  
Vol 85 (9) ◽  
Author(s):  
Erin B. Purcell ◽  
Robert W. McKee ◽  
David S. Courson ◽  
Elizabeth M. Garrett ◽  
Shonna M. McBride ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Stationary Phase ◽  
Nutrient Availability ◽  
Biofilm Formation ◽  
Biochemical Characterization ◽  
Toxin Production ◽  
Physiological Adaptation ◽  
Cyclic Diguanylate ◽  
Content Type ◽  
Wide Range

ABSTRACT The signaling molecule cyclic diguanylate (c-di-GMP) mediates physiological adaptation to extracellular stimuli in a wide range of bacteria. The complex metabolic pathways governing c-di-GMP synthesis and degradation are highly regulated, but the specific cues that impact c-di-GMP signaling are largely unknown. In the intestinal pathogen Clostridium difficile, c-di-GMP inhibits flagellar motility and toxin production and promotes pilus-dependent biofilm formation, but no specific biological functions have been ascribed to any of the individual c-di-GMP synthases or phosphodiesterases (PDEs). Here, we report the functional and biochemical characterization of a c-di-GMP PDE, PdcA, 1 of 37 confirmed or putative c-di-GMP metabolism proteins in C. difficile 630. Our studies reveal that pdcA transcription is controlled by the nutrient-regulated transcriptional regulator CodY and accordingly increases during stationary phase. In addition, PdcA PDE activity is allosterically regulated by GTP, further linking c-di-GMP levels to nutrient availability. Mutation of pdcA increased biofilm formation and reduced toxin biosynthesis without affecting swimming motility or global intracellular c-di-GMP. Analysis of the transcriptional response to pdcA mutation indicates that PdcA-dependent phenotypes manifest during stationary phase, consistent with regulation by CodY. These results demonstrate that inactivation of this single PDE gene is sufficient to impact multiple c-di-GMP-dependent phenotypes, including the production of major virulence factors, and suggest a link between c-di-GMP signaling and nutrient availability.

scholarly journals Evaluating the Effects of Surotomycin Treatment on Clostridium difficile Toxin A and B Production, Immune Response, and Morphological Changes

2016 ◽  
Vol 60 (6) ◽  
pp. 3519-3523 ◽  
Author(s):  
Bradley T. Endres ◽  
Eugénie Bassères ◽  
Mohammed Khaleduzzaman ◽  
M. Jahangir Alam ◽  
Laurent Chesnel ◽  
...  
Keyword(s):  
Immune Response ◽  
Clostridium Difficile ◽  
Morphological Changes ◽  
Toxin Production ◽  
Morphological Data ◽  
Growth Media ◽  
Toxin A ◽  
Content Type ◽  
Cyclic Lipopeptide ◽  
The Impact

Surotomycin is a cyclic lipopeptide in development forClostridium difficile-associated diarrhea. This study aimed to assess the impact of surotomycin exposure onC. difficiletoxin A and B concentrations and the associated changes in immune response in comparison to vancomycin and metronidazole. Time-kill curve assays were performed using strain R20291 (BI/NAP1/027) at supra-MICs (4× and 40×) and sub-MICs (0.5×) of surotomycin and comparators. Following treatment, CFU counts, toxin A and B concentrations, and cellular morphological changes using scanning electron microscopy were examined. Inflammatory response was determined by measuring interleukin-8 (IL-8) concentrations from polarized Caco-2 cells exposed to antibiotic-treatedC. difficilegrowth media. Supra-MICs (4× and 40×) of surotomycin resulted in a reduction of vegetative cells over 72 h (4-log difference,P< 0.01) compared to controls. These results correlated with decreases of 77% and 68% in toxin A and B production at 48 h, respectively (P< 0.005, each), which resulted in a significant reduction in IL-8 concentration compared to controls. Similar results were observed with comparator antibiotics. Bacterial cell morphology showed that the cell wall was broken apart by surotomycin treatment at supra-MICs while sub-MIC studies showed a “deflated” phenotype plus a rippling effect. These results suggest that surotomycin has potent killing effects onC. difficilethat results in reduced toxin production and attenuates the immune response similar to comparator antibiotics. The morphological data also confirm observations that surotomycin is a membrane-active antibiotic.

scholarly journals Effect of Ceftobiprole Treatment on Growth of and Toxin Production by Clostridium difficile in Cecal Contents of Mice

2011 ◽  
Vol 55 (5) ◽  
pp. 2174-2177 ◽  
Author(s):  
Michelle M. Nerandzic ◽  
Curtis J. Donskey
Keyword(s):  
Clostridium Difficile ◽  
Inhibitory Activity ◽  
Toxin Production ◽  
Content Type ◽  
Anaerobic Microflora

ABSTRACTCeftobiprole and ceftobiprole medocaril did not promote growth of or toxin production byClostridium difficilein mouse cecal contents, whereas ceftazidime, cefoxitin, ceftriaxone, cefotaxime, and ertapenem did. The relatively low propensity of ceftobiprole to promoteC. difficilewas attributable to inhibitory activity againstC. difficileand sparing of anaerobic microflora.

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