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Bile salt inhibition of host cell damage by Clostridium difficile toxins.

Darkoh C, Brown EL, Kaplan HB, DuPont HL - PLoS ONE (2013)

Bottom Line: Remarkably, addition of 5 mM taurocholate reduced caspase-3 activity in cells treated with 2, 4, 6, and 12 µg/ml of toxin B by 99%, 78%, 64%, and 60%, respectively.Furthermore, spent culture medium from Caco-2 cells incubated with both toxin B and taurocholate exhibited significantly decreased lactate dehydrogenase activity compared to spent culture medium from cells incubated with toxin B only.Our results suggest that the mechanism of taurocholate-mediated inhibition functions at the level of toxin activity since taurocholate did not affect C. difficile growth and toxin production.

View Article: PubMed Central - PubMed

Affiliation: The University of Texas School of Public Health, Division of Epidemiology, Human Genetics and Environmental Sciences, Center For Infectious Diseases, Houston, Texas, United States of America ; The University of Texas Graduate School of Biomedical Sciences, Houston, Texas, United States of America.

ABSTRACT
Virulent Clostridium difficile strains produce toxin A and/or toxin B that are the etiological agents of diarrhea and pseudomembranous colitis. Treatment of C. difficile infections (CDI) has been hampered by resistance to multiple antibiotics, sporulation, emergence of strains with increased virulence, recurrence of the infection, and the lack of drugs that preserve or restore the colonic bacterial flora. As a result, there is new interest in non-antibiotic CDI treatments. The human conjugated bile salt taurocholate was previously shown in our laboratory to inhibit C. difficile toxin A and B activities in an in vitro assay. Here we demonstrate for the first time in an ex vivo assay that taurocholate can protect Caco-2 colonic epithelial cells from the damaging effects of the C. difficile toxins. Using caspase-3 and lactate dehydrogenase assays, we have demonstrated that taurocholate reduced the extent of toxin B-induced apoptosis and cell membrane damage. Confluent Caco-2 cells cultured with toxin B induced elevated caspase-3 activity. Remarkably, addition of 5 mM taurocholate reduced caspase-3 activity in cells treated with 2, 4, 6, and 12 µg/ml of toxin B by 99%, 78%, 64%, and 60%, respectively. Furthermore, spent culture medium from Caco-2 cells incubated with both toxin B and taurocholate exhibited significantly decreased lactate dehydrogenase activity compared to spent culture medium from cells incubated with toxin B only. Our results suggest that the mechanism of taurocholate-mediated inhibition functions at the level of toxin activity since taurocholate did not affect C. difficile growth and toxin production. These findings open up a new avenue for the development of non-antibiotic therapeutics for CDI treatment.

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Taurocholate significantly decreases C. difficile toxin B-mediated induction of caspase-3 activity in Caco-2 cells.Confluent Caco-2 cells were incubated for 24 hrs with 0, 4, 8, 12 and 24 µg of toxin B in the presence (+) or absence (−) of 5 mM taurocholate. Cell monolayers were scraped from the 24-well plates and lysed to obtain crude protein lysates. Crude protein lysates (75 µg) were incubated with the caspase-3 colorimetric substrate reagent (Invitrogen) for 8 hrs at 37°C and absorbance at 410 nm was measured. A molar extinction coefficient for p-nitrophenol of ε = 17700 M−1cm−1 was used in the calculations of caspace-3 activity [56]. One-Way ANOVA test showed a significant difference (P<0.003) between the caspase-3 activities of the cells cultured with 5 mM taurocholate and the cells cultured without taurocholate. Error bars represents the standard deviation from three replicate experiments.
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pone-0079631-g003: Taurocholate significantly decreases C. difficile toxin B-mediated induction of caspase-3 activity in Caco-2 cells.Confluent Caco-2 cells were incubated for 24 hrs with 0, 4, 8, 12 and 24 µg of toxin B in the presence (+) or absence (−) of 5 mM taurocholate. Cell monolayers were scraped from the 24-well plates and lysed to obtain crude protein lysates. Crude protein lysates (75 µg) were incubated with the caspase-3 colorimetric substrate reagent (Invitrogen) for 8 hrs at 37°C and absorbance at 410 nm was measured. A molar extinction coefficient for p-nitrophenol of ε = 17700 M−1cm−1 was used in the calculations of caspace-3 activity [56]. One-Way ANOVA test showed a significant difference (P<0.003) between the caspase-3 activities of the cells cultured with 5 mM taurocholate and the cells cultured without taurocholate. Error bars represents the standard deviation from three replicate experiments.

Mentions: During C. difficile infection, apoptosis is an important downstream effect resulting from receptor-mediated toxin endocytosis into the host cell cytoplasm and subsequent inactivation of the host GTPases. Previous reports showed that toxin A induces cell death in human epithelial cells ex vivo via the activation of caspases [58], [59], [60]. Our results indicate that various amounts of toxin B (4, 8, 12, and 24 µg) induced significant caspase-3 activity in Caco-2 cells (Fig. 3). The caspase-3 activation cascade plays a crucial role in several apoptotic mechanisms, including activation of key apoptotic mediators essential to chromatin condensation, DNA fragmentation, dismantling of the cell, and formation of apoptotic bodies [61], [62], [63]. To determine whether taurocholate protected the Caco-2 cells from toxin B damage by preventing apoptosis, caspase-3 activity was assessed in taurocholate-treated and untreated cells. Crude protein lysates prepared from confluent Caco-2 cells incubated with various amounts of toxin B in the presence or absence of taurocholate were tested for caspase-3 activity. In cells cultured without toxin B or taurocholate, no caspase-3 activity was detected. In the presence of toxin B, however, caspase-3 activity was detected in a dose-dependent manner (Fig. 3). Cells cultured with 4–24 µg of toxin B showed a 2.5-7.5-fold increase in caspase-3 activity. In contrast, cells cultured with toxin B in the presence of taurocholate had significant reduction in caspase-3 activity. Remarkably, addition of 5 mM taurocholate reduced caspase-3 activity in cells treated with 4, 8, 12, and 24 µg of toxin B by 99%, 78%, 64%, and 60%, respectively. These data demonstrated that taurocholate protected Caco-2 cells from the damaging effects of toxin B, as evidenced by the decreased levels of the pro-apoptotic protease, caspase-3.


Bile salt inhibition of host cell damage by Clostridium difficile toxins.

Darkoh C, Brown EL, Kaplan HB, DuPont HL - PLoS ONE (2013)

Taurocholate significantly decreases C. difficile toxin B-mediated induction of caspase-3 activity in Caco-2 cells.Confluent Caco-2 cells were incubated for 24 hrs with 0, 4, 8, 12 and 24 µg of toxin B in the presence (+) or absence (−) of 5 mM taurocholate. Cell monolayers were scraped from the 24-well plates and lysed to obtain crude protein lysates. Crude protein lysates (75 µg) were incubated with the caspase-3 colorimetric substrate reagent (Invitrogen) for 8 hrs at 37°C and absorbance at 410 nm was measured. A molar extinction coefficient for p-nitrophenol of ε = 17700 M−1cm−1 was used in the calculations of caspace-3 activity [56]. One-Way ANOVA test showed a significant difference (P<0.003) between the caspase-3 activities of the cells cultured with 5 mM taurocholate and the cells cultured without taurocholate. Error bars represents the standard deviation from three replicate experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3823588&req=5

pone-0079631-g003: Taurocholate significantly decreases C. difficile toxin B-mediated induction of caspase-3 activity in Caco-2 cells.Confluent Caco-2 cells were incubated for 24 hrs with 0, 4, 8, 12 and 24 µg of toxin B in the presence (+) or absence (−) of 5 mM taurocholate. Cell monolayers were scraped from the 24-well plates and lysed to obtain crude protein lysates. Crude protein lysates (75 µg) were incubated with the caspase-3 colorimetric substrate reagent (Invitrogen) for 8 hrs at 37°C and absorbance at 410 nm was measured. A molar extinction coefficient for p-nitrophenol of ε = 17700 M−1cm−1 was used in the calculations of caspace-3 activity [56]. One-Way ANOVA test showed a significant difference (P<0.003) between the caspase-3 activities of the cells cultured with 5 mM taurocholate and the cells cultured without taurocholate. Error bars represents the standard deviation from three replicate experiments.
Mentions: During C. difficile infection, apoptosis is an important downstream effect resulting from receptor-mediated toxin endocytosis into the host cell cytoplasm and subsequent inactivation of the host GTPases. Previous reports showed that toxin A induces cell death in human epithelial cells ex vivo via the activation of caspases [58], [59], [60]. Our results indicate that various amounts of toxin B (4, 8, 12, and 24 µg) induced significant caspase-3 activity in Caco-2 cells (Fig. 3). The caspase-3 activation cascade plays a crucial role in several apoptotic mechanisms, including activation of key apoptotic mediators essential to chromatin condensation, DNA fragmentation, dismantling of the cell, and formation of apoptotic bodies [61], [62], [63]. To determine whether taurocholate protected the Caco-2 cells from toxin B damage by preventing apoptosis, caspase-3 activity was assessed in taurocholate-treated and untreated cells. Crude protein lysates prepared from confluent Caco-2 cells incubated with various amounts of toxin B in the presence or absence of taurocholate were tested for caspase-3 activity. In cells cultured without toxin B or taurocholate, no caspase-3 activity was detected. In the presence of toxin B, however, caspase-3 activity was detected in a dose-dependent manner (Fig. 3). Cells cultured with 4–24 µg of toxin B showed a 2.5-7.5-fold increase in caspase-3 activity. In contrast, cells cultured with toxin B in the presence of taurocholate had significant reduction in caspase-3 activity. Remarkably, addition of 5 mM taurocholate reduced caspase-3 activity in cells treated with 4, 8, 12, and 24 µg of toxin B by 99%, 78%, 64%, and 60%, respectively. These data demonstrated that taurocholate protected Caco-2 cells from the damaging effects of toxin B, as evidenced by the decreased levels of the pro-apoptotic protease, caspase-3.

Bottom Line: Remarkably, addition of 5 mM taurocholate reduced caspase-3 activity in cells treated with 2, 4, 6, and 12 µg/ml of toxin B by 99%, 78%, 64%, and 60%, respectively.Furthermore, spent culture medium from Caco-2 cells incubated with both toxin B and taurocholate exhibited significantly decreased lactate dehydrogenase activity compared to spent culture medium from cells incubated with toxin B only.Our results suggest that the mechanism of taurocholate-mediated inhibition functions at the level of toxin activity since taurocholate did not affect C. difficile growth and toxin production.

View Article: PubMed Central - PubMed

Affiliation: The University of Texas School of Public Health, Division of Epidemiology, Human Genetics and Environmental Sciences, Center For Infectious Diseases, Houston, Texas, United States of America ; The University of Texas Graduate School of Biomedical Sciences, Houston, Texas, United States of America.

ABSTRACT
Virulent Clostridium difficile strains produce toxin A and/or toxin B that are the etiological agents of diarrhea and pseudomembranous colitis. Treatment of C. difficile infections (CDI) has been hampered by resistance to multiple antibiotics, sporulation, emergence of strains with increased virulence, recurrence of the infection, and the lack of drugs that preserve or restore the colonic bacterial flora. As a result, there is new interest in non-antibiotic CDI treatments. The human conjugated bile salt taurocholate was previously shown in our laboratory to inhibit C. difficile toxin A and B activities in an in vitro assay. Here we demonstrate for the first time in an ex vivo assay that taurocholate can protect Caco-2 colonic epithelial cells from the damaging effects of the C. difficile toxins. Using caspase-3 and lactate dehydrogenase assays, we have demonstrated that taurocholate reduced the extent of toxin B-induced apoptosis and cell membrane damage. Confluent Caco-2 cells cultured with toxin B induced elevated caspase-3 activity. Remarkably, addition of 5 mM taurocholate reduced caspase-3 activity in cells treated with 2, 4, 6, and 12 µg/ml of toxin B by 99%, 78%, 64%, and 60%, respectively. Furthermore, spent culture medium from Caco-2 cells incubated with both toxin B and taurocholate exhibited significantly decreased lactate dehydrogenase activity compared to spent culture medium from cells incubated with toxin B only. Our results suggest that the mechanism of taurocholate-mediated inhibition functions at the level of toxin activity since taurocholate did not affect C. difficile growth and toxin production. These findings open up a new avenue for the development of non-antibiotic therapeutics for CDI treatment.

Show MeSH
Related in: MedlinePlus