Clostridium difficile Toxin A Induces Intestinal Epithelial Cell Apoptosis and Damage: Role of Gln and Ala-Gln in Toxin A Effects

Brito, Gerly A.C.; Carneiro‐Filho, Benedito A.; Oriá, Reinaldo B.; Destura, Raul V.; Lima, Aldo A. M.; Guerrant, Richard L.

doi:10.1007/s10620-005-2771-x

Cited by 43 publications

(54 citation statements)

References 38 publications

(39 reference statements)

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The GTD has been shown to target and inactivate a number of Rho-family GTPases (19,20). This inactivation has been linked to a cell rounding or cytopathic effect (CPE) (21)(22)(23)(24) and to an apoptotic cytotoxic effect (25)(26)(27)(28)(29)(30)(31)(32)(33). In tissue culture models, the apoptotic effects of TcdA and TcdB occur at toxin concentrations of picomolar or lower and are evident at 24 to 48 h postintoxication (26,28,29,(34)(35)(36).…”

mentioning

confidence: 99%

Clostridium difficile Toxins TcdA and TcdB Cause Colonic Tissue Damage by Distinct Mechanisms

et al. 2016

View full text Add to dashboard Cite

e As the major cause of antibiotic-associated diarrhea, Clostridium difficile is a serious problem in health care facilities worldwide. C. difficile produces two large toxins, TcdA and TcdB, which are the primary virulence factors in disease. The respective functions of these toxins have been difficult to discern, in part because the cytotoxicity profiles for these toxins differ with concentration and cell type. The goal of this study was to develop a cell culture model that would allow a side-by-side mechanistic comparison of the toxins. Conditionally immortalized, young adult mouse colonic (YAMC) epithelial cells demonstrate an exquisite sensitivity to both toxins with phenotypes that agree with observations in tissue explants. TcdA intoxication results in an apoptotic cell death that is dependent on the glucosyltransferase activity of the toxin. In contrast, TcdB has a bimodal mechanism; it induces apoptosis in a glucosyltransferase-dependent manner at lower concentrations and glucosyltransferase-independent necrotic death at higher concentrations. The direct comparison of the responses to TcdA and TcdB in cells and colonic explants provides the opportunity to unify a large body of observations made by many independent investigators. C lostridium difficile is the most common cause of antibioticassociated diarrhea in the United States, and C. difficile infection (CDI) has been steadily increasing in prevalence and severity over the last 15 years (1-3). Symptoms of CDI can range from mild diarrhea to pseudomembranous colitis, and hallmarks of the disease include neutrophil infiltration, fluid release, and necrotic lesions in the colonic epithelium (4, 5). The bacteria produce two main virulence factors, large toxins called TcdA and TcdB (6, 7).The respective function and relative importance of each toxin in pathogenesis have been active topics of investigation. Genetic knockout experiments in C. difficile have shown that both toxins are important for disease pathology, although TcdB alone is sufficient to cause death in both the hamster and mouse models (6-8). For many years, TcdA and TcdB have been thought to act synergistically, with TcdA acting as an enterotoxin and TcdB acting as a cytotoxin (9, 10). The general term enterotoxin refers to the capacity of TcdA to induce inflammation, cytokine release, and fluid secretion in animal intoxication models (11-13). While TcdB does not always induce these same phenotypes in models, such as the ileal loop model, it has been shown to disrupt the integrity of the epithelial structure in human explant and xenograft models (14, 15). TcdB is also notably more potent as a cytotoxin in cell culture models (9, 10, 16).The toxins have an N-terminal glucosyltransferase domain (GTD) that is delivered into the host cytosol by the C-terminal portion of the protein (17, 18). The GTD has been shown to target and inactivate a number of Rho-family GTPases (19,20). This inactivation has been linked to a cell rounding or cytopathic effect (CPE) (21-24) and to an apoptotic cytotoxic ef...

show abstract

mentioning

confidence: 99%

Clostridium difficile Toxins TcdA and TcdB Cause Colonic Tissue Damage by Distinct Mechanisms

et al. 2016

View full text Add to dashboard Cite

show abstract

“…Conventionally, cell susceptibility to bacterial toxins and neutralizing antibody responses in vitro rely on radioactive cytotoxicity measurements for protein synthesis or are evaluated by microscopic observations of intoxicated cell monolayers. These methods may be subjective, time-consuming, and inherently low throughput, due to the requirements of manual observation and counting of cells (5,7,9,(19)(20)(21)(22). Because of these limitations, we sought to develop an alternative assay that may be used as a versatile platform for measuring bacterial toxin activity and to evaluate the immunogenicities of toxin-based vaccines.…”

mentioning

confidence: 99%

Caspase Activation as a Versatile Assay Platform for Detection of Cytotoxic Bacterial Toxins

et al. 2013

View full text Add to dashboard Cite

Pathogenic bacteria produce several virulence factors that help them establish infection in permissive hosts. Bacterial toxins are a major class of virulence factors and hence are attractive therapeutic targets for vaccine development. Here, we describe the development of a rapid, sensitive, and high-throughput assay that can be used as a versatile platform to measure the activities of bacterial toxins. We have exploited the ability of these toxins to cause cell death via apoptosis of sensitive cultured cell lines as a readout for measuring toxin activity. Caspases (cysteine-aspartic proteases) are induced early in the apoptotic pathway, and so we used their induction to measure the activities of Clostridium difficile toxins A (TcdA) and B (TcdB) and binary toxin (CDTaCDTb), Corynebacterium diphtheriae toxin (DT), and Pseudomonas aeruginosa exotoxin A (PEA). Caspase induction in the cell lines, upon exposure to toxins, was optimized by toxin concentration and intoxication time, and the specificity of caspase activity was established using a genetically mutated toxin and a pan-caspase inhibitor. In addition, we demonstrate the utility of the caspase assay for measuring toxin potency, as well as neutralizing antibody (NAb) activity against C. difficile toxins. Furthermore, the caspase assay showed excellent correlation with the filamentous actin (F-actin) polymerization assay for measuring TcdA and TcdB neutralization titers upon vaccination of hamsters. These results demonstrate that the detection of caspase induction due to toxin exposure using a chemiluminescence readout can support potency and clinical immunogenicity testing for bacterial toxin vaccine candidates in development.

show abstract

“…Although a trend toward reduction of apoptosis was seen, we could not show a significant glutamine effect in our in vitro model. Other studies, including our own [46,47], have found a protective role of glutamine in preventing cell apoptosis seen with toxins, atrophy or cytokines [47][48][49] in other more differentiated intestinal cell lines. However, we postulate that apoptosis at the level of undifferentiated stem cells is critical to the effects of 5-FU in preventing the emergence of mutant cell lines in the crypts, which would prevent further neoplasic growth.…”

Section: Discussionmentioning

confidence: 51%

“…The optimal dose of 10 mM of glutamine and alanyl-glutamine show enhanced crypt cell proliferation following 5-FU challenge. The same concentration proved to be beneficial in our previous studies [47,51] with Clostridium difficile toxin A-induced epithelial injury. This concentration used in the medium far exceeds the physiological serum concentration, confirming the high requirement for these peptides for cell growth and adaptation following severe epithelial injury.…”

Section: Discussionmentioning

confidence: 68%

Alanyl-Glutamine and Glutamine Supplementation Improves 5-Fluorouracil-Induced Intestinal Epithelium Damage In Vitro

et al. 2008

Self Cite

View full text Add to dashboard Cite

In this study, we have examined the role of glutamine derivatives in reducing 5-fluorouracil (5-FU)-induced epithelial damage in an undifferentiated crypt intestinal cell line, IEC-6. In this model, we have investigated proliferation indirectly by detecting the enzyme-derived formazan dye from the tetrazolium salt WST-1 in viable cells at 24 and 48 h after 5-FU treatment. Migration was measured at 12 and 24 h after razor scraping of the cell monolayer. Cell death was measured by quantifying the percentage of apoptotic and necrotic figures by flow cytometry at 12 and 24 h following 5-FU challenge. Neither glutamine nor alanyl-glutamine prevented 5-FU-induced apoptosis and necrosis in IEC-6 cells at 12 and 24 h after 5-FU challenge. However, glutamine and alanyl-glutamine enhanced migration and proliferation when compared with 5-FU-treated controls (P <0.05). These new findings support our earlier study on the benefit of oral glutamine in enhancing epithelial recovery after 5-FU challenge.

show abstract

Clostridium difficile Toxin A Induces Intestinal Epithelial Cell Apoptosis and Damage: Role of Gln and Ala-Gln in Toxin A Effects

Cited by 43 publications

References 38 publications

Clostridium difficile Toxins TcdA and TcdB Cause Colonic Tissue Damage by Distinct Mechanisms

Clostridium difficile Toxins TcdA and TcdB Cause Colonic Tissue Damage by Distinct Mechanisms

Caspase Activation as a Versatile Assay Platform for Detection of Cytotoxic Bacterial Toxins

Alanyl-Glutamine and Glutamine Supplementation Improves 5-Fluorouracil-Induced Intestinal Epithelium Damage In Vitro

Contact Info

Product

Resources

About