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Clostridial glucosylating toxins enter cells via clathrin-mediated endocytosis.

Papatheodorou P, Zamboglou C, Genisyuerek S, Guttenberg G, Aktories K - PLoS ONE (2010)

Bottom Line: We found that the intoxication of cultured cells by CGTs was strongly delayed when cells were preincubated with dynasore, a cell-permeable inhibitor of dynamin, or chlorpromazine, an inhibitor of the clathrin-dependent endocytic pathway.In addition, lipid rafts impairment by exogenous depletion of sphingomyelin did not decelerate intoxication of HeLa cells by CGTs.Taken together, our data indicate that the endocytic uptake of the CGTs involves a dynamin-dependent process that is mainly governed by clathrin.

View Article: PubMed Central - PubMed

Affiliation: Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany.

ABSTRACT
Clostridium difficile toxin A (TcdA) and toxin B (TcdB), C. sordellii lethal toxin (TcsL) and C. novyi alpha-toxin (TcnA) are important pathogenicity factors, which represent the family of the clostridial glucosylating toxins (CGTs). Toxin A and B are associated with antibiotic-associated diarrhea and pseudomembraneous colitis. Lethal toxin is involved in toxic shock syndrome after abortion and alpha-toxin in gas gangrene development. CGTs enter cells via receptor-mediated endocytosis and require an acidified endosome for translocation of the catalytic domain into the cytosol. Here we studied the endocytic processes that mediate cell internalization of the CGTs. Intoxication of cells was monitored by analyzing cell morphology, status of Rac glucosylation in cell lysates and transepithelial resistance of cell monolayers. We found that the intoxication of cultured cells by CGTs was strongly delayed when cells were preincubated with dynasore, a cell-permeable inhibitor of dynamin, or chlorpromazine, an inhibitor of the clathrin-dependent endocytic pathway. Additional evidence about the role of clathrin in the uptake of the prototypical CGT family member toxin B was achieved by expression of a dominant-negative inhibitor of the clathrin-mediated endocytosis (Eps15 DN) or by siRNA against the clathrin heavy chain. Accordingly, cells that expressed dominant-negative caveolin-1 were not protected from toxin B-induced cell rounding. In addition, lipid rafts impairment by exogenous depletion of sphingomyelin did not decelerate intoxication of HeLa cells by CGTs. Taken together, our data indicate that the endocytic uptake of the CGTs involves a dynamin-dependent process that is mainly governed by clathrin.

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Disintegration of lipid rafts by exogenous sphingomyelin depletion with SMase.HeLa cells were pretreated with SMase or left untreated prior to intoxication with (A) 4 pM toxin B for 75 min, 5 nM toxin A for 300 min, 5 nM lethal toxin for 180 min, 5 nM α-toxin for 120 min, (B) mock incubation for 300 min, or (C) 50 nM VacA toxin for 300 min. Images were obtained by microscopy, upon onset of intoxication characteristics. (D) SMase-preincubated or non-preincubated HeLa cells were intoxicated with 4 pM toxin B or left untreated. The percentage of cell rounding was quantified from three independent experiments at indicated time points (data are given +/− SD).
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pone-0010673-g005: Disintegration of lipid rafts by exogenous sphingomyelin depletion with SMase.HeLa cells were pretreated with SMase or left untreated prior to intoxication with (A) 4 pM toxin B for 75 min, 5 nM toxin A for 300 min, 5 nM lethal toxin for 180 min, 5 nM α-toxin for 120 min, (B) mock incubation for 300 min, or (C) 50 nM VacA toxin for 300 min. Images were obtained by microscopy, upon onset of intoxication characteristics. (D) SMase-preincubated or non-preincubated HeLa cells were intoxicated with 4 pM toxin B or left untreated. The percentage of cell rounding was quantified from three independent experiments at indicated time points (data are given +/− SD).

Mentions: Caveolae-mediated endocytic processes are exclusively taking place at microdomains of the plasma membrane denoted as lipid rafts [30]. But there is growing evidence for the implication of lipid rafts also in clathrin-dependent uptake mechanisms [31],[32],[33]. Therefore, we wanted to study the role of lipid rafts in the uptake of the CGTs. Sphingomyelin is a prominent component of lipid rafts and can be exogenously depleted from cell membranes by the addition of the enzyme sphingomyelinase (SMase) from Bacillus cereus [34]. In HeLa cells pretreated with SMase, no significant delay in cell rounding was observed upon addition of toxin B, toxin A, lethal toxin or α-toxin, when compared with non-pretreated cells (Fig. 5A). Control cells that were incubated with SMase alone did not show morphological alterations when compared with untreated cells (Fig. 5B). The exogenous depletion of sphingomyelin in HeLa cells was evaluated by using the vacuolating cytotoxin A (VacA) from Helicobacter pylori as a marker for sphingomyelin-/lipid rafts-dependent entry into host cells [35],[36]. As expected, vacuolization in HeLa cells upon addition of VacA was strongly reduced in sphingomyelin-depleted cells, when compared with control cells under equal conditions (85% reduction) (Fig. 5C). The lack of protection of HeLa cells from toxin B-induced cell rounding by SMase pretreatment was further confirmed in a time-dependent manner (Fig. 5D). In conclusion, lipid rafts seem not to be crucially involved in processes leading to the endocytic uptake of CGTs.


Clostridial glucosylating toxins enter cells via clathrin-mediated endocytosis.

Papatheodorou P, Zamboglou C, Genisyuerek S, Guttenberg G, Aktories K - PLoS ONE (2010)

Disintegration of lipid rafts by exogenous sphingomyelin depletion with SMase.HeLa cells were pretreated with SMase or left untreated prior to intoxication with (A) 4 pM toxin B for 75 min, 5 nM toxin A for 300 min, 5 nM lethal toxin for 180 min, 5 nM α-toxin for 120 min, (B) mock incubation for 300 min, or (C) 50 nM VacA toxin for 300 min. Images were obtained by microscopy, upon onset of intoxication characteristics. (D) SMase-preincubated or non-preincubated HeLa cells were intoxicated with 4 pM toxin B or left untreated. The percentage of cell rounding was quantified from three independent experiments at indicated time points (data are given +/− SD).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2871790&req=5

pone-0010673-g005: Disintegration of lipid rafts by exogenous sphingomyelin depletion with SMase.HeLa cells were pretreated with SMase or left untreated prior to intoxication with (A) 4 pM toxin B for 75 min, 5 nM toxin A for 300 min, 5 nM lethal toxin for 180 min, 5 nM α-toxin for 120 min, (B) mock incubation for 300 min, or (C) 50 nM VacA toxin for 300 min. Images were obtained by microscopy, upon onset of intoxication characteristics. (D) SMase-preincubated or non-preincubated HeLa cells were intoxicated with 4 pM toxin B or left untreated. The percentage of cell rounding was quantified from three independent experiments at indicated time points (data are given +/− SD).
Mentions: Caveolae-mediated endocytic processes are exclusively taking place at microdomains of the plasma membrane denoted as lipid rafts [30]. But there is growing evidence for the implication of lipid rafts also in clathrin-dependent uptake mechanisms [31],[32],[33]. Therefore, we wanted to study the role of lipid rafts in the uptake of the CGTs. Sphingomyelin is a prominent component of lipid rafts and can be exogenously depleted from cell membranes by the addition of the enzyme sphingomyelinase (SMase) from Bacillus cereus [34]. In HeLa cells pretreated with SMase, no significant delay in cell rounding was observed upon addition of toxin B, toxin A, lethal toxin or α-toxin, when compared with non-pretreated cells (Fig. 5A). Control cells that were incubated with SMase alone did not show morphological alterations when compared with untreated cells (Fig. 5B). The exogenous depletion of sphingomyelin in HeLa cells was evaluated by using the vacuolating cytotoxin A (VacA) from Helicobacter pylori as a marker for sphingomyelin-/lipid rafts-dependent entry into host cells [35],[36]. As expected, vacuolization in HeLa cells upon addition of VacA was strongly reduced in sphingomyelin-depleted cells, when compared with control cells under equal conditions (85% reduction) (Fig. 5C). The lack of protection of HeLa cells from toxin B-induced cell rounding by SMase pretreatment was further confirmed in a time-dependent manner (Fig. 5D). In conclusion, lipid rafts seem not to be crucially involved in processes leading to the endocytic uptake of CGTs.

Bottom Line: We found that the intoxication of cultured cells by CGTs was strongly delayed when cells were preincubated with dynasore, a cell-permeable inhibitor of dynamin, or chlorpromazine, an inhibitor of the clathrin-dependent endocytic pathway.In addition, lipid rafts impairment by exogenous depletion of sphingomyelin did not decelerate intoxication of HeLa cells by CGTs.Taken together, our data indicate that the endocytic uptake of the CGTs involves a dynamin-dependent process that is mainly governed by clathrin.

View Article: PubMed Central - PubMed

Affiliation: Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany.

ABSTRACT
Clostridium difficile toxin A (TcdA) and toxin B (TcdB), C. sordellii lethal toxin (TcsL) and C. novyi alpha-toxin (TcnA) are important pathogenicity factors, which represent the family of the clostridial glucosylating toxins (CGTs). Toxin A and B are associated with antibiotic-associated diarrhea and pseudomembraneous colitis. Lethal toxin is involved in toxic shock syndrome after abortion and alpha-toxin in gas gangrene development. CGTs enter cells via receptor-mediated endocytosis and require an acidified endosome for translocation of the catalytic domain into the cytosol. Here we studied the endocytic processes that mediate cell internalization of the CGTs. Intoxication of cells was monitored by analyzing cell morphology, status of Rac glucosylation in cell lysates and transepithelial resistance of cell monolayers. We found that the intoxication of cultured cells by CGTs was strongly delayed when cells were preincubated with dynasore, a cell-permeable inhibitor of dynamin, or chlorpromazine, an inhibitor of the clathrin-dependent endocytic pathway. Additional evidence about the role of clathrin in the uptake of the prototypical CGT family member toxin B was achieved by expression of a dominant-negative inhibitor of the clathrin-mediated endocytosis (Eps15 DN) or by siRNA against the clathrin heavy chain. Accordingly, cells that expressed dominant-negative caveolin-1 were not protected from toxin B-induced cell rounding. In addition, lipid rafts impairment by exogenous depletion of sphingomyelin did not decelerate intoxication of HeLa cells by CGTs. Taken together, our data indicate that the endocytic uptake of the CGTs involves a dynamin-dependent process that is mainly governed by clathrin.

Show MeSH
Related in: MedlinePlus