Release of TcdA and TcdB from Clostridium difficile cdi 630 is not affected by functional inactivation of the tcdE gene

Olling, Alexandra; Seehase, Sophie; Minton, Nigel P.; Tatge, Helma; Schröter, Saskia; Kohlscheen, Saskia; Pich, Andreas; Just, Ingo; Gerhard, Ralf

doi:10.1016/j.micpath.2011.10.009

Cited by 63 publications

(64 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Stringent criteria for final protein identification were used to reduce potential falsepositive identification, as described in Materials and Methods. In total, about 350 proteins were detected in supernatants of strain 630⌬erm, a number comparable to that reported in an earlier secretome analysis of 630⌬erm (32).…”

Section: Analyses Of Protein Secretome In Ty Brothsupporting

confidence: 81%

“…While toxin production has been shown to increase during the stationary phase of growth (12,20,32), genes involved in motility are expressed at higher levels during the exponential phase of growth (36). Correspondingly, we chose to examine toxin production in flagellar mutants during the logarithmic phase of growth, where flagellar gene expression and assembly of the flagellar apparatus are optimal.…”

Section: Generation Of Flagellar Mutant Strainsmentioning

confidence: 99%

“…TcdR is an alternative sigma factor required for transcription of the two toxin genes; TcdE has been described to be a putative holin-like protein involved in toxin secretion, although this role has recently been a source of debate (17,32); and TcdC is an anti-sigma factor that negatively regulates tcdR-dependent transcription (28,29). In addition, four other regulators of toxin synthesis have recently been identified: CepA (3), CodY (10), SpoOA (42), and SigH (36).…”

mentioning

confidence: 99%

See 2 more Smart Citations

Modulation of Toxin Production by the Flagellar Regulon in Clostridium difficile

et al. 2012

View full text Add to dashboard Cite

We show in this study that toxin production in Clostridium difficile is altered in cells which can no longer form flagellar filaments. The impact of inactivation of fliC, CD0240, fliF, fliG, fliM, and flhB-fliR flagellar genes upon toxin levels in culture supernatants was assessed using cell-based cytotoxicity assay, proteomics, immunoassay, and immunoblotting approaches. Each of these showed that toxin levels in supernatants were significantly increased in a fliC mutant compared to that in the C. difficile 630 parent strain. In contrast, the toxin levels in supernatants secreted from other flagellar mutants were significantly reduced compared with that in the parental C. difficile 630 strain. Transcriptional analysis of the pathogenicity locus genes (tcdR, tcdB, tcdE, and tcdA) revealed a significant increase of all four genes in the fliC mutant strain, while transcription of all four genes was significantly reduced in fliM, fliF, fliG, and flhB-fliR mutants. These results demonstrate that toxin transcription in C. difficile is modulated by the flagellar regulon. More significantly, mutant strains showed a corresponding change in virulence compared to the 630 parent strain when tested in a hamster model of C. difficile infection. This is the first demonstration of differential flagellum-related transcriptional regulation of toxin production in C. difficile and provides evidence for elaborate regulatory networks for virulence genes in C. difficile. Clostridium difficile is a Gram-positive spore-forming bacillus which is recognized to be the major cause of nosocomial diarrhea associated with antibiotic therapy (35). The incidence of C. difficile infection has been rapidly increasing in both Europe and North America, and this increase in infections has been associated with a significantly high mortality rate (2, 35). The broad spectrum of diseases caused by C. difficile, which range from antibiotic-associated diarrhea to the potentially lethal, pseudomembranous colitis, has been shown to depend on the level of toxin produced (1), and this production is recognized as a critical determinant of pathogenicity. Following antibiotic therapy when the microbiota of the gastrointestinal tract is disrupted, infection by C. difficile is mediated by spores which germinate in the gut, followed by vegetative cell proliferation and the subsequent secretion of the two major virulence factors, the Rho glucosylating toxins TcdA and TcdB.The toxin-encoding genes (tcdA and tcdB) are localized to a 19.6-kb pathogenicity locus (PaLoc) which includes three other accessory genes, tcdR, tcdE and tcdC (43). TcdR is an alternative sigma factor required for transcription of the two toxin genes; TcdE has been described to be a putative holin-like protein involved in toxin secretion, although this role has recently been a source of debate (17, 32); and TcdC is an anti-sigma factor that negatively regulates tcdR-dependent transcription (28, 29). In addition, four other regulators of toxin synthesis have recently been identified: CepA (3), CodY ...

show abstract

Section: Analyses Of Protein Secretome In Ty Brothsupporting

confidence: 81%

Section: Generation Of Flagellar Mutant Strainsmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Modulation of Toxin Production by the Flagellar Regulon in Clostridium difficile

et al. 2012

View full text Add to dashboard Cite

show abstract

“…Following SDS-PAGE, bands on the polyacrylamide gel containing proteins of interest were excised. Proteins were digested with trypsin and subjected to mass spectrometry (MS) analysis by following the protocols of Olling and colleagues (37), with some modifications. MS and tandem MS (MS-MS) spectra were acquired in positive ion mode with a scan range of m/z 300 to 1600 using a top10 method and collision-induced dissociation (CID) fragmentation.…”

Section: Methodsmentioning

confidence: 99%

The LasB Elastase of Pseudomonas aeruginosa Acts in Concert with Alkaline Protease AprA To Prevent Flagellin-Mediated Immune Recognition

et al. 2016

View full text Add to dashboard Cite

eThe opportunistic pathogen Pseudomonas aeruginosa is capable of establishing severe and persistent infections in various eukaryotic hosts. It encodes a wide array of virulence factors and employs several strategies to evade immune detection. In the present study, we screened the Harvard Medical School transposon mutant library of P. aeruginosa PA14 for bacterial factors that modulate interleukin-8 responses in A549 human airway epithelial cells. We found that in addition to the previously identified alkaline protease AprA, the elastase LasB is capable of degrading exogenous flagellin under calcium-replete conditions and prevents flagellin-mediated immune recognition. Our results indicate that the production of two proteases with anti-flagellin activity provides a failsafe mechanism for P. aeruginosa to ensure the maintenance of protease-dependent immune-modulating functions.

show abstract

“…Given tcdE's close localization to tcdB and tcdA on the PaLoc and the reported ability of TcdE to lyse Escherichia coli when expressed as a recombinant protein, the concept has merit. Yet this idea has come under more scrutiny recently with the publication of two diametrically opposed reports, one of which found no role for TcdE in toxin release (89), while the other showed TcdE to be absolutely essential for toxin release from the organism (90). Both studies relied on in vitro analyses.…”

Section: Factors Important To C Difficile Virulencementioning

confidence: 99%

Variations in Virulence and Molecular Biology among Emerging Strains of Clostridium difficile

Hunt

Ballard

2013

Microbiol Mol Biol Rev

View full text Add to dashboard Cite

SUMMARY Clostridium difficile is a Gram-positive, spore-forming organism which infects and colonizes the large intestine, produces potent toxins, triggers inflammation, and causes significant systemic complications. Treating C. difficile infection (CDI) has always been difficult, because the disease is both caused and resolved by antibiotic treatment. For three and a half decades, C. difficile has presented a treatment challenge to clinicians, and the situation took a turn for the worse about 10 years ago. An increase in epidemic outbreaks related to CDI was first noticed around 2003, and these outbreaks correlated with a sudden increase in the mortality rate of this illness. Further studies discovered that these changes in CDI epidemiology were associated with the rapid emergence of hypervirulent strains of C. difficile , now collectively referred to as NAP1/BI/027 strains. The discovery of new epidemic strains of C. difficile has provided a unique opportunity for retrospective and prospective studies that have sought to understand how these strains have essentially replaced more historical strains as a major cause of CDI. Moreover, detailed studies on the pathogenesis of NAP1/BI/027 strains are leading to new hypotheses on how this emerging strain causes severe disease and is more commonly associated with epidemics. In this review, we provide an overview of CDI, discuss critical mechanisms of C. difficile virulence, and explain how differences in virulence-associated factors between historical and newly emerging strains might explain the hypervirulence exhibited by this pathogen during the past decade.

show abstract

Release of TcdA and TcdB from Clostridium difficile cdi 630 is not affected by functional inactivation of the tcdE gene

Cited by 63 publications

References 22 publications

Modulation of Toxin Production by the Flagellar Regulon in Clostridium difficile

Modulation of Toxin Production by the Flagellar Regulon in Clostridium difficile

The LasB Elastase of Pseudomonas aeruginosa Acts in Concert with Alkaline Protease AprA To Prevent Flagellin-Mediated Immune Recognition

Variations in Virulence and Molecular Biology among Emerging Strains of Clostridium difficile

Contact Info

Product

Resources

About