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Clostridium difficile toxin A attenuates Wnt/?-catenin signaling in intestinal epithelial cells.

ABSTRACT: Clostridium difficile toxins A and B (TcdA and TcdB) are homologous glycosyltransferases that inhibit a group of small GTPases within host cells, but several mechanisms underlying their pathogenic activity remain unclear. In this study, we evaluated the effects of TcdA on the Wnt/?-catenin pathway, the major driving force behind the proliferation of epithelial cells in colonic crypts. IEC-6 and RKO cells stimulated with Wnt3a-conditioned medium were incubated with 10, 50, and 100 ng/ml of TcdA for 24 h, resulting in a dose-dependent inhibition of the Wnt signaling, as demonstrated by a T-cell factor (TCF) reporter assay. This was further confirmed by immunofluorescence staining for nuclear localization of ?-catenin and Western blotting for ?-catenin and c-Myc (encoded by a Wnt target gene). Moreover, our Western blot analysis showed a decrease in the ?-catenin protein levels, which was reversed by z-VAD-fmk, a pan-caspase inhibitor. Nonetheless, TcdA was still able to inhibit the Wnt/?-catenin pathway even in the presence of z-VAD-fmk, lithium chloride (a GSK3? inhibitor), or constitutively active ?-catenin, as determined by a TCF reporter assay. Furthermore, preincubation of RKO cells with TcdA for 12 h also attenuated Wnt3a-mediated activation of Wnt signaling, suggesting that inactivation of Rho GTPases plays a significant role in that inhibition. Taken together, these findings suggest that attenuation of the Wnt signaling by TcdA is important for TcdA antiproliferative effects.

SUBMITTER: Bezerra Lima B

PROVIDER: S-EPMC4097644 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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Clostridium difficile toxin A attenuates Wnt/β-catenin signaling in intestinal epithelial cells.

Bezerra Lima Bruno B Faria Fonseca Bárbara B da Graça Amado Nathália N Moreira Lima Débora D Albuquerque Ribeiro Ronaldo R Garcia Abreu José J de Castro Brito Gerly Anne GA

Infection and immunity 20140407 7

Clostridium difficile toxins A and B (TcdA and TcdB) are homologous glycosyltransferases that inhibit a group of small GTPases within host cells, but several mechanisms underlying their pathogenic activity remain unclear. In this study, we evaluated the effects of TcdA on the Wnt/β-catenin pathway, the major driving force behind the proliferation of epithelial cells in colonic crypts. IEC-6 and RKO cells stimulated with Wnt3a-conditioned medium were incubated with 10, 50, and 100 ng/ml of TcdA f ...[more]

PMID: 24711571

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Project description:UNLABELLED:Clostridium difficile infection (CDI) is dramatically increasing as a cause of antibiotic- and hospital-associated diarrhea worldwide. C. difficile, a multidrug-resistant pathogen, flourishes in the colon after the gut microbiota has been altered by antibiotic therapy. Consequently, it produces toxins A and B that directly cause disease. Despite the enormous public health problem posed by this pathogen, the molecular mechanisms that regulate production of the toxins, which are directly responsible for disease, remained largely unknown until now. Here, we show that C. difficile toxin synthesis is regulated by an accessory gene regulator quorum-signaling system, which is mediated through a small (<1,000-Da) thiolactone that can be detected directly in stools of CDI patients. These findings provide direct evidence of the mechanism of regulation of C. difficile toxin synthesis and offer exciting new avenues both for rapid detection of C. difficile infection and development of quorum-signaling-based non-antibiotic therapies to combat this life-threatening emerging pathogen. IMPORTANCE:Clostridium difficile infection (CDI) is the most common definable cause of hospital-acquired and antibiotic-associated diarrhea in the United States, with the total cost of treatment estimated between 1 and 4.8 billion U.S. dollars annually. C. difficile, a Gram-positive, spore-forming anaerobe, flourishes in the colon after the gut microbiota has been altered by antibiotic therapy. As a result, there is an urgent need for non-antibiotic CDI treatments that preserve the colonic microbiota. C. difficile produces toxins A and B, which are directly responsible for disease. Here, we report that C. difficile regulates its toxin synthesis by quorum signaling, in which a novel signaling peptide activates transcription of the disease-causing toxin genes. This finding provides new therapeutic targets to be harnessed for novel nonantibiotic therapy for C. difficile infections.

Dataset Information

Clostridium difficile toxin A attenuates Wnt/?-catenin signaling in intestinal epithelial cells.

Publications

Clostridium difficile toxin A attenuates Wnt/β-catenin signaling in intestinal epithelial cells.

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