Clostridial Glucosylating Toxins Enter Cells via Clathrin-Mediated Endocytosis

Clostridium difficile toxin A (TcdA) and toxin B (TcdB), C. sordellii lethal toxin (TcsL) and C. novyi α-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 α-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

Accumulation of FlAsH/Lumio Green in active mitochondria can be reversed by β-mercaptoethanol for specific staining of tetracysteine-tagged proteins

Recent advances in the field of small molecule labels for live cell imaging promise to overcome some of the limitations set by the size of fluorescent proteins. We tested the tetracysteine–biarsenical labeling system in live cell fluorescence microscopy of reggie-1/flotillin-2 in HeLa and N2a cells. In both cell types, the biarsenical staining reagent FlAsH/Lumio Green accumulated in active mitochondria and led to mitochondrial swelling. This is indicative of toxic side effects caused by arsenic, which should be considered when this labeling system is to be used in live cell imaging. Mitochondrial accumulation of FlAsH/Lumio Green was reversed by addition of low concentrations of thiol-containing reagents during labeling and a subsequent high stringency thiol wash. Both ethanedithiol and β-mercaptoethanol proved to be effective. We therefore established a staining protocol using β-mercaptoethanol as thiol binding site competitor resulting in a specific staining of tetracysteine-tagged reggie-1/flotillin-2 of adequate signal to noise ratio, so that the more toxic and inconvenient ethanedithiol could be avoided. Furthermore, we show that staining efficiency was greatly enhanced by introducing a second tetracysteine sequence in tandem

RNAi-mediated gene silencing of the clathrin heavy chain.

<p>HeLa cells were transfected with siRNA against the clathrin heavy chain (si_clathrin). (A) Two days after transfection, cell lysates from si_clathrin-transfected cells and mock-transfected cells were analyzed for clathrin heavy chain expression using a specific antibody (α-clathrin(HC)). Antibody detection of the housekeeping protein glyceraldehyde 3-phosphate dehydrogenase (α-GAPDH) served as a control for equal loading of lysate samples. (B) After addition of toxin B (4 pM), Rac1 glucosylation status was compared in lysates from si_clathrin- and mock-transfected HeLa cells at indicated time points with antibodies recognizing either only unmodified Rac1 (α-Rac1_non-Glc) or all Rac1 (α-Rac1_total).</p

Expression of dominant-negative Eps15 and Cav-1.

<p>(A) HeLa cells, transfected with plasmids, encoding dominant-negative forms of Eps15 or Cav-1 fused to a GFP moiety, were eventually intoxicated for 75 min with 4 pM toxin B or left untreated. Toxin B-induced alterations in cell morphology were analyzed in Eps15- (Eps15 DN::EGFP), Cav-1- (GFP::Cav-1 DN) and mock-transfected cells after actin staining with FITC-phalloidin (red and grey signals) and by applying fluorescence microscopy. Green signals derived from cells expressing GFP-tagged, dominant-negative Eps15 or Cav-1, respectively. (B) HeLa cells were transfected with a plasmid encoding Eps15 DN::EGFP, intoxicated with 4 pM toxin B for 30 min and subsequently subjected to fluorescence-assisted cell sorting. GFP excitation of the Eps15 DN::EGFP-transfected cells resulted in two cell populations (left panel, blue curve, bordered with dashed line). One population represents non-transfected cells, with overlapping background fluorescence as obtained in mock-transfected cells (left panel, red curve). The other population represents Eps15 DN::EGFP-expressing cells. Equal number of cells from both cell populations (non-transf. and Eps15 DN::EGFP) where subjected to cell lysis and analysis of the Rac1 glucosylation status with antibodies recognizing either only unmodified Rac1 (α-Rac1_non-Glc) or all Rac1 (α-Rac1_total). (C) Procedure was performed essentially as described in (B), but with HeLa cells transfected with a plasmid encoding GFP::Cav-1.</p

Disintegration of lipid rafts by exogenous sphingomyelin depletion with SMase.

<p>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).</p

Pharmacological inhibition of clathrin assembly with chlorpromazine.

<p>HeLa cells were preincubated with chlorpromazine (Cp) or left untreated, prior to addition of (A) 5 nM diphtheria toxin (DT) and incubation for 180 min, (B) 3.5 nM CNF1 and incubation for 150 min or (C) 4 pM toxin B and incubation for 75 min. (D) The percentage of cell rounding in chlorpromazine-pretreated or non-pretreated HeLa cells after intoxication with 4 pM toxin B for 75 min, 1.5 nM toxin A for 105 min, 4 nM lethal toxin for 75 min or 1.5 nM α-toxin for 60 min was quantified and data are given +/− SD (n = 3) and from a minimum of 200 cells in total. (E) Human intestinal epithelial cells (CaCo-2) were grown to confluency on filters and were preincubated with chlorpromazine (Cp) or left untreated. A subset of cells was intoxicated with 40 pM toxin B and transepithelial electrical resistance (TER) was measured at indicated time points, where starting resistance was set to 100% and TER values are calculated as relative TER in % from starting resistance (+/− SD, n = 3).</p

Inhibition of dynamin with dynasore and a dominant-negative mutant.

<p>Dynasore-pretreated and non-pretreated HeLa cells were incubated with (A) 5 nM diphtheria toxin for 180 min or (B) 4 pM toxin B for 75 min, prior to microscopical analysis of the cell morphology. (C) HeLa cells preincubated with dynasore or with solvent only were treated with 4 pM toxin B for 75 min, 1.5 nM toxin A for 105 min, 4 nM lethal toxin for 75 min or 1.5 nM α-toxin for 60 min. The percentage of rounded cells was quantified and data are given +/− SD (n = 3) and from a minimum of 200 cells in total. (D) Dynasore-pretreated HT-29 cells and untreated cells were intoxicated with 40 pM toxin. After onset of cell rounding (150 min), glucosylation status of Rac1 in cell lysates was analyzed with antibodies recognizing either only unmodified Rac1 (α-Rac1_non-Glc) or all Rac1 (α-Rac1_total). (E) HeLa cells expressing dominant-negative dynamin (HA-dynamin_K44A) for 24 h or mock-transfected cells were intoxicated with 4 pM toxin B for 75 min and cell morphology analyzed microscopically. (F) Selective expression of HA-dynamin_K44A in plasmid-transfected HeLa cells, but not in mock-transfected cells, was detected in cell lysates with an anti-HA antibody. Antibody detection of the housekeeping protein glyceraldehyde 3-phosphate dehydrogenase (α-GAPDH) served as a control for equal loading of lysate samples.</p