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 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 c-di-GMP Inhibits Early Sporulation in Clostridioides difficile

mSphere ◽  
2021 ◽  
Author(s):  
Adrianne N. Edwards ◽  
Caitlin L. Willams ◽  
Nivedita Pareek ◽  
Shonna M. McBride ◽  
Rita Tamayo
Keyword(s):  
Life Cycle ◽  
Biofilm Formation ◽  
Toxin Production ◽  
Cyclic Diguanylate ◽  
Content Type ◽  
Critical Step ◽  
Clostridioides Difficile ◽  
Physiological Processes ◽  
Gastrointestinal Pathogen ◽  
The Impact

Many bacterial organisms utilize the small signaling molecule cyclic diguanylate (c-di-GMP) to regulate important physiological processes, including motility, toxin production, biofilm formation, and colonization. c-di-GMP inhibits motility and toxin production and promotes biofilm formation and colonization in the anaerobic, gastrointestinal pathogen Clostridioides difficile . However, the impact of c-di-GMP on C. difficile spore formation, a critical step in this pathogen’s life cycle, is unknown.

scholarly journals The Phosphotransfer Protein CD1492 Represses Sporulation Initiation in Clostridium difficile

2016 ◽  
Vol 84 (12) ◽  
pp. 3434-3444 ◽  
Author(s):  
Kevin O. Childress ◽  
Adrianne N. Edwards ◽  
Kathryn L. Nawrocki ◽  
Sarah E. Anderson ◽  
Emily C. Woods ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Clostridium Difficile ◽  
Molecular Mechanisms ◽  
Toxin Production ◽  
Negative Regulator ◽  
Hamster Model ◽  
Content Type ◽  
Sporulation Initiation

The formation of spores is critical for the survival ofClostridium difficileoutside the host gastrointestinal tract. Persistence ofC. difficilespores greatly contributes to the spread ofC. difficileinfection (CDI), and the resistance of spores to antimicrobials facilitates the relapse of infection. Despite the importance of sporulation toC. difficilepathogenesis, the molecular mechanisms controlling spore formation are not well understood. The initiation of sporulation is known to be regulated through activation of the conserved transcription factor Spo0A. Multiple regulators influence Spo0A activation in other species; however, many of these factors are not conserved inC. difficileand few novel factors have been identified. Here, we investigated the function of a protein, CD1492, that is annotated as a kinase and was originally proposed to promote sporulation by directly phosphorylating Spo0A. We found that deletion ofCD1492resulted in increased sporulation, indicating that CD1492 is a negative regulator of sporulation. Accordingly, we observed increased transcription of Spo0A-dependent genes in theCD1492mutant. Deletion of CD1492 also resulted in decreased toxin productionin vitroand in decreased virulence in the hamster model of CDI. Further, theCD1492mutant demonstrated effects on gene expression that are not associated with Spo0A activation, including lowersigDandrstAtranscription, suggesting that this protein interacts with factors other than Spo0A. Altogether, the data indicate that CD1492 negatively affects sporulation and positively influences motility and virulence. These results provide further evidence thatC. difficilesporulation is regulated differently from that of other endospore-forming species.

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 Regulation of Type IV Pili Contributes to Surface Behaviors of Historical and Epidemic Strains of Clostridium difficile

2015 ◽  
Vol 198 (3) ◽  
pp. 565-577 ◽  
Author(s):  
Erin B. Purcell ◽  
Robert W. McKee ◽  
Eric Bordeleau ◽  
Vincent Burrus ◽  
Rita Tamayo
Keyword(s):  
Clostridium Difficile ◽  
Biofilm Formation ◽  
The United States ◽  
Type Iv Pili ◽  
Flagellar Motility ◽  
Content Type ◽  
Type Iv ◽  
Intestinal Pathogen ◽  
Surface Motility ◽  
Pilin Gene

ABSTRACTThe intestinal pathogenClostridium difficileis an urgent public health threat that causes antibiotic-associated diarrhea and is a leading cause of fatal nosocomial infections in the United States.C. difficilerates of recurrence and mortality have increased in recent years due to the emergence of so-called “hypervirulent” epidemic strains. A great deal of the basic biology ofC. difficilehas not been characterized. Recent findings that flagellar motility, toxin synthesis, and type IV pilus (TFP) formation are regulated by cyclic diguanylate (c-di-GMP) reveal the importance of this second messenger forC. difficilegene regulation. However, the function(s) of TFP inC. difficileremains largely unknown. Here, we examine TFP-dependent phenotypes and the role of c-di-GMP in controlling TFP production in the historical 630 and epidemic R20291 strains ofC. difficile. We demonstrate that TFP contribute toC. difficilebiofilm formation in both strains, but with a more prominent role in R20291. Moreover, we report that R20291 is capable of TFP-dependent surface motility, which has not previously been described inC. difficile. The expression and regulation of thepilA1pilin gene differs between R20291 and 630, which may underlie the observed differences in TFP-mediated phenotypes. The differences inpilA1expression are attributable to greater promoter-driven transcription in R20291. In addition, R20291, but not 630, upregulates c-di-GMP levels during surface-associated growth, suggesting that the bacterium senses its substratum. The differential regulation of surface behaviors in historical and epidemicC. difficilestrains may contribute to the different infection outcomes presented by these strains.IMPORTANCEHowClostridium difficileestablishes and maintains colonization of the host bowel is poorly understood. Surface behaviors ofC. difficileare likely relevant during infection, representing possible interactions between the bacterium and the intestinal environment. Pili mediate bacterial interactions with various surfaces and contribute to the virulence of many pathogens. We report that type IV pili (TFP) contribute to biofilm formation byC. difficile. TFP are also required for surface motility, which has not previously been demonstrated forC. difficile. Furthermore, an epidemic-associatedC. difficilestrain showed higher pilin gene expression and greater dependence on TFP for biofilm production and surface motility. Differences in TFP regulation and their effects on surface behaviors may contribute to increased virulence in recent epidemic strains.

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 Specific Control of Pseudomonas aeruginosa Surface-Associated Behaviors by Two c-di-GMP Diguanylate Cyclases

mBio ◽  
2010 ◽  
Vol 1 (4) ◽  
Author(s):  
Judith H. Merritt ◽  
Dae-Gon Ha ◽  
Kimberly N. Cowles ◽  
Wenyun Lu ◽  
Diana K. Morales ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Extracellular Polysaccharide ◽  
Diguanylate Cyclase ◽  
Flagellar Motility ◽  
Critical Question ◽  
Cyclic Diguanylate ◽  
Critical Signal ◽  
Content Type ◽  
Wide Range

ABSTRACT The signaling nucleotide cyclic diguanylate (c-di-GMP) regulates the transition between motile and sessile growth in a wide range of bacteria. Understanding how microbes control c-di-GMP metabolism to activate specific pathways is complicated by the apparent multifold redundancy of enzymes that synthesize and degrade this dinucleotide, and several models have been proposed to explain how bacteria coordinate the actions of these many enzymes. Here we report the identification of a diguanylate cyclase (DGC), RoeA, of Pseudomonas aeruginosa that promotes the production of extracellular polysaccharide (EPS) and contributes to biofilm formation, that is, the transition from planktonic to surface-dwelling cells. Our studies reveal that RoeA and the previously described DGC SadC make distinct contributions to biofilm formation, controlling polysaccharide production and flagellar motility, respectively. Measurement of total cellular levels of c-di-GMP in ∆roeA and ∆sadC mutants in two different genetic backgrounds revealed no correlation between levels of c-di-GMP and the observed phenotypic output with regard to swarming motility and EPS production. Our data strongly argue against a model wherein changes in total levels of c-di-GMP can account for the specific surface-related phenotypes of P. aeruginosa. IMPORTANCE A critical question in the study of cyclic diguanylate (c-di-GMP) signaling is how the bacterial cell integrates contributions of multiple c-di-GMP-metabolizing enzymes to mediate its cognate functional outputs. One leading model suggests that the effects of c-di-GMP must, in part, be localized subcellularly. The data presented here show that the phenotypes controlled by two different diguanylate cyclase (DGC) enzymes have discrete outputs despite the same total level of c-di-GMP. These data support and extend the model in which localized c-di-GMP signaling likely contributes to coordination of the action of the multiple proteins involved in the synthesis, degradation, and/or binding of this critical signal.

scholarly journals Indole Acts as an Extracellular Cue Regulating Gene Expression in Vibrio cholerae

2009 ◽  
Vol 191 (11) ◽  
pp. 3504-3516 ◽  
Author(s):  
Ryan S. Mueller ◽  
Sinem Beyhan ◽  
Simran G. Saini ◽  
Fitnat H. Yildiz ◽  
Douglas H. Bartlett
Keyword(s):  
Gene Expression ◽  
Vibrio Cholerae ◽  
Biofilm Formation ◽  
Signal Molecule ◽  
Control Of Gene Expression ◽  
Protozoan Grazing ◽  
Extracellular Signal ◽  
Grazing Resistance ◽  
Indole Signaling

ABSTRACT Indole has been proposed to act as an extracellular signal molecule influencing biofilm formation in a range of bacteria. For this study, the role of indole in Vibrio cholerae biofilm formation was examined. It was shown that indole activates genes involved in vibrio polysaccharide (VPS) production, which is essential for V. cholerae biofilm formation. In addition to activating these genes, it was determined using microarrays that indole influences the expression of many other genes, including those involved in motility, protozoan grazing resistance, iron utilization, and ion transport. A transposon mutagenesis screen revealed additional components of the indole-VPS regulatory circuitry. The indole signaling cascade includes the DksA protein along with known regulators of VPS production, VpsR and CdgA. A working model is presented in which global control of gene expression by indole is coordinated through σ54 and associated transcriptional regulators.

scholarly journals Cyclic Di-GMP Regulates Multiple Cellular Functions in the Symbiotic Alphaproteobacterium Sinorhizobium meliloti

2015 ◽  
Vol 198 (3) ◽  
pp. 521-535 ◽  
Author(s):  
Simon Schäper ◽  
Elizaveta Krol ◽  
Dorota Skotnicka ◽  
Volkhard Kaever ◽  
Rolf Hilker ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Sinorhizobium Meliloti ◽  
Acid Stress ◽  
Second Messenger ◽  
Lifestyle Changes ◽  
Single Domain ◽  
Receptor Protein ◽  
Leguminous Plant ◽  
Content Type

ABSTRACTSinorhizobium melilotiundergoes major lifestyle changes between planktonic states, biofilm formation, and symbiosis with leguminous plant hosts. In many bacteria, the second messenger 3′,5′-cyclic di-GMP (c-di-GMP, or cdG) promotes a sessile lifestyle by regulating a plethora of processes involved in biofilm formation, including motility and biosynthesis of exopolysaccharides (EPS). Here, we systematically investigated the role of cdG inS. melilotiRm2011 encoding 22 proteins putatively associated with cdG synthesis, degradation, or binding. Single mutations in 21 of these genes did not cause evident changes in biofilm formation, motility, or EPS biosynthesis. In contrast, manipulation of cdG levels by overproducing endogenous or heterologous diguanylate cyclases (DGCs) or phosphodiesterases (PDEs) affected these processes and accumulation ofN-Acyl-homoserine lactones in the culture supernatant. Specifically, individual overexpression of theS. melilotigenespleD,SMb20523,SMb20447,SMc01464, andSMc03178encoding putative DGCs and ofSMb21517encoding a single-domain PDE protein had an impact and resulted in increased levels of cdG. Compared to the wild type, anS. melilotistrain that did not produce detectable levels of cdG (cdG0) was more sensitive to acid stress. However, it was symbiotically potent, unaffected in motility, and only slightly reduced in biofilm formation. TheSMc01790-SMc01796locus, homologous to theAgrobacterium tumefaciensuppABCDEFcluster governing biosynthesis of a unipolarly localized polysaccharide, was found to be required for cdG-stimulated biofilm formation, while the single-domain PilZ protein McrA was identified as a cdG receptor protein involved in regulation of motility.IMPORTANCEWe present the first systematic genome-wide investigation of the role of 3′,5′-cyclic di-GMP (c-di-GMP, or cdG) in regulation of motility, biosynthesis of exopolysaccharides, biofilm formation, quorum sensing, and symbiosis in a symbiotic alpha-rhizobial species. Phenotypes of anS. melilotistrain unable to produce cdG (cdG0) demonstrated that this second messenger is not essential for root nodule symbiosis but may contribute to acid tolerance. Our data further suggest that enhanced levels of cdG promote sessility ofS. melilotiand uncovered a single-domain PilZ protein as regulator of motility.

scholarly journals Biofilm Formation Caused by Clinical Acinetobacter baumannii Isolates Is Associated with Overexpression of the AdeFGH Efflux Pump

2015 ◽  
Vol 59 (8) ◽  
pp. 4817-4825 ◽  
Author(s):  
Xinlong He ◽  
Feng Lu ◽  
Fenglai Yuan ◽  
Donglin Jiang ◽  
Peng Zhao ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Gene Transcription ◽  
Biofilm Formation ◽  
Efflux Pump ◽  
Outer Membrane Proteins ◽  
Content Type ◽  
Potential Association ◽  
Genes Encoding ◽  
Clinical Environments

ABSTRACTChronic wound infections are associated with biofilm formation, which in turn has been correlated with drug resistance. However, the mechanism by which bacteria form biofilms in clinical environments is not clearly understood. This study was designed to investigate the biofilm formation potency ofAcinetobacter baumanniiand the potential association of biofilm formation with genes encoding efflux pumps, quorum-sensing regulators, and outer membrane proteins. A total of 48 clinically isolatedA. baumanniistrains, identified by enterobacterial repetitive intergenic consensus (ERIC)-PCR as types A-II, A-III, and A-IV, were analyzed. Three representative strains, which were designatedA. baumanniiABR2, ABR11, and ABS17, were used to evaluate antimicrobial susceptibility, biofilm inducibility, and gene transcription (abaI,adeB,adeG,adeJ,carO, andompA). A significant increase in the MICs of different classes of antibiotics was observed in the biofilm cells. The formation of a biofilm was significantly induced in all the representative strains exposed to levofloxacin. The levels of gene transcription varied between bacterial genotypes, antibiotics, and antibiotic concentrations. The upregulation ofadeGcorrelated with biofilm induction. The consistent upregulation ofadeGandabaIwas detected in A-III-typeA. baumanniiin response to levofloxacin and meropenem (1/8 to 1/2× the MIC), conditions which resulted in the greatest extent of biofilm induction. This study demonstrates a potential role of the AdeFGH efflux pump in the synthesis and transport of autoinducer molecules during biofilm formation, suggesting a link between low-dose antimicrobial therapy and a high risk of biofilm infections caused byA. baumannii. This study provides useful information for the development of antibiofilm strategies.

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