1. Introduction
Clostridium difficile (
C. difficile) causes enteric diseases in patients treated with broad-spectrum antibiotics that range from diarrhea to severe, potentially life-threatening pseudomembranous colitis because disturbance of the gut flora enables spore germination and growth of this pathogen [
1]. The causative agents of
C. difficile-associated diseases are the exotoxins A (TcdA, 308 kDa) and B (TcdB, 270 kDa), which catalyse the glucosylation of Rho, Rac and Cdc42 in the cytosol of cells thereby inhibiting signal transduction via these GTPases [
2,
3]. This action leads to destruction of the actin cytoskeleton, cell rounding and loss of integrity of the intestinal wall (for review see [
4]). In addition to toxins A and B, about 6%–35% of the strains produce the binary actin ADP-ribosylating toxin CDT [
5,
6,
7], which directly attacks the actin cytoskeleton and contributes to the hypervirulence of these strains with associated increased patients morbidity and mortality [
7,
8,
9,
10,
11,
12,
13]. Like the other members of the clostridial binary actin-ADP-ribosylating toxins family,
C. botulinum C2 toxin [
14,
15,
16],
C. perfringens iota toxin [
17,
18,
19,
20], and
C. spiroforme transferase (CST) [
21], CDT consists of two non-linked proteins, which must assemble on the surface of target cells to exhibit their cytotoxic effects (for review see [
22,
23]). The binding/translocation component CDTb binds to lipolysis stimulated receptor (LSR), which is the protein receptor for CDT, CST and iota toxin [
24,
25] and induces clustering of LSR in lipid rafts [
26]. Besides LSR, CD44 is involved in binding of CDT and the other iota-like toxins to target cells and might serve as a co-receptor [
27]. After uptake of the CDTb/CDTa complexes by receptor-mediated endocytosis, CDTa translocates from acidified endosomes into the cytosol [
28] to ADP-ribosylate G-actin [
5,
29]. The molecular and cellular consequences following toxin-catalysed mono-ADP-ribosylation of actin at arginine-177 were described in detail for the related C2 and iota toxins [
14,
30,
31,
32,
33,
34,
35,
36,
37]. Taken together, this modification inhibits actin polymerization [
38] and causes cell-rounding. Moreover, it also affects the microtubules, which form long protrusions around the cell body and in the case of CDT it was shown that these protrusions bind
C. difficile and increase its adherence to enterocytes [
39,
40].
We provided evidence that the transport of CDTa across endosomal membranes into the cytosol occurs by a pH- and chaperone-dependent translocation mechanism [
28], which seems to be common for the binary clostridial actin ADP-ribosylating toxins and was previously investigated for the C2 and iota toxins in more detail [
41,
42]. After proteolytic activation, the binding/translocation components of these toxins, C2IIa and Ib, respectively, form heptamers, which bind to their cellular receptors and assemble with the enzyme components C2I and Ia, respectively [
41,
42,
43,
44,
45,
46,
47]. After receptor-mediated endocytosis of the toxin complexes, the binding/translocation components mediate the translocation of the enzyme components from the lumen of acidified endosomal vesicles into the cytosol [
28,
41,
42,
48,
49]. To this end, the binding/translocation components change their conformation due to the acidic conditions, insert into the endosomal membranes and form trans-membrane pores [
41,
42,
48,
50,
51,
52,
53,
54]. These pores serve as translocation channels for the unfolded enzyme components and are essential prerequisites for their transport across endosomal membranes into the cytosol [
48,
53,
55], which is in analogy with the anthrax toxin PA
63 channel [
56]. In addition to the pores, cytosolic host cell factors including chaperones and protein folding helper enzymes are involved in membrane translocation of the enzyme components of C2 toxin [
57,
58], iota toxin [
28,
59] and CDT [
28].
Due to their essential role in toxin uptake, the translocation pores represent attractive molecular drug targets [
60] to protect cells from these binary toxins. We and others identified pore blockers for C2 toxin and iota toxin, but also for the related binary anthrax toxin (for review see [
61,
62,
63]), such as small-molecule positively charged aromatic compounds [
64,
65,
66,
67,
68] and tailored β-cyclodextrin derivatives [
69,
70,
71,
72,
73,
74,
75,
76,
77,
78] and characterized their inhibitory effects on the transmembrane pores formed by these toxins
in vitro and in living cells. The tailored seven-fold symmetrical positively charged per-6-
S-(3-aminomethyl)benzylthio-β-cyclodextrin (AMBnTβ-CD, see
Figure 1D) efficiently blocks PA
63, the translocation pore of anthrax toxin and prevents intoxication with anthrax toxin
in vitro, in intact cells and in animal models [
69,
79]. Recently, we demonstrated that AMBnTβ-CD is also a potent pore blocker for C2IIa and Ib [
74,
76]. AMBnTβ-CD protects cultured cells from intoxication with C2 and iota toxins by inhibiting the channel-mediated membrane translocation of C2I and Ib [
76]. Since the closely related binding/translocation components of CDT and iota toxin are functionally interchangeable [
80] and exploit the same receptor on target cells [
24,
25], here we investigate whether AMBnTβ-CD also inhibits translocation of CDTa and protects cells from intoxication with CDT.
3. Experimental Section
3.1. Materials and Reagents
Cell culture medium MEM and fetal calf serum were purchased from Invitrogen (Karlsruhe, Germany) and cell culture materials from TPP (Trasadingen, Switzerland). Complete
® protease inhibitor was from Roche (Mannheim, Germany), the protein molecular weight marker Page Ruler prestained Protein ladder
® from Fermentas (St. Leon-Rot, Germany), biotin-labelled NAD
+ from R & D Systems GmbH (Wiesbaden-Nordenstadt, Germany), bafilomycin A1 from Calbiochem (Bad Soden, Germany). AMBnTβ-CD was custom synthesized at LycloLab (Budapest, Hungary) as described in detail previously (compound 14b, [
70]). CDTa and CDTb (from
C. difficile strain 196) were expressed as recombinant His-tagged proteins in the
B. megaterium expression system and purified as described earlier [
24].
3.2. Cell Culture and Intoxication Assays
African green monkey kidney (Vero) cells were cultivated at 37 °C and 5% CO
2 in MEM containing 10% heat-inactivated fetal calf serum, 1.5 g/L sodium bicarbonate, 1 mM sodium-pyruvate, 2 mM L-glutamine, 0.1 mM non-essential amino acids and 10 mg/mL Penicillin/Streptomycin. Vero cells were trypsinized and reseeded twice a week for at most 15–20 times. For cytotoxicity experiments, cells grown in culture dishes in serum-free medium were incubated at 37 °C with CDT and after the indicated incubation periods visualized by using a Zeiss Axiovert 40CFl microscope (Oberkochen, Germany) with a Jenoptik progress C10 CCD camera (Carl Zeiss GmbH, Jena, Germany). The CDT-induced cell rounding as specific indication of the intoxication process and inhibitory effects of AMBnTβ-CD were analysed by incubating cells with CDT in the presence and absence of this substance. The percentage of round cells was determined from the pictures. The pH-induced translocation of cell-bound CDT across the cytoplasmic membrane of Vero cells was performed as described earlier [
28].
3.3. Quantification of F-Actin Content in Cells
Vero cells grown in a 96-well plate were pre-treated for 30 min at 37 °C with AMBnTβ-CD or left untreated for control. Then, cells were incubated for further 2 h with CDT. As an additional control, cells were left untreated. Subsequently, the medium was removed and cells were fixed by 20 min incubation with paraformaldehyde (4% in PBS) and permeabilized with Triton-X100 (0.4% in PBS). Non-specific binding sites were blocked by incubating the cells for 45 min with 5% non-fat dry milk in PBS containing 0.1% Tween-20 (PBS-T) and F-actin was stained by 45 min incubation at 37 °C with phalloidin-FITC. Cells were washed and the fluorescence measured at 513 nm emission with a TecanReader Infinite M1000 (Tecan Germany, Crailsheim, Germany).
3.4. SDS-PAGE and Western Blotting
For the Western blot analysis of cell-bound CDTb, cells were incubated for 30 min at 4 °C with CDTb in the presence (10 min pre-treatment) or absence of AMBnTβ-CD, washed and lysed. Equal amounts of lysate protein were subjected to SDS-PAGE according to the method of Laemmli [
82] and blotted onto a nitrocellulose membrane (Whatman, Dassel, Germany). The membrane was blocked for 30 min with 5% non-fat dry milk in PBS-T and probed with a specific antibody against iota b (a kind gift from Bradley G. Stiles, Integrated Toxicology, Bacteriology Divisions, U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD, USA), which cross-reacts with the closely related CDTb. The membrane was washed with PBS-T, incubated with anti-rabbit antibody coupled to horseradish peroxidase (Santa-Cruz, Heidelberg, Germany), washed again, and CDTb was detected with the enhanced chemiluminescence (ECL) system from Millipore (Schwalbach, Germany) according to the manufacturer’s instructions.
3.5. ADP-Ribosylation of Actin by CDTa in a Cell-Free System
Vero cell lysate (10 µg of protein) was pre-incubated for 10 min at 37 °C together with the inhibitor AMBnTβ-CD or left untreated for control. Subsequently, 500 ng/mL of CDTa and 10 µM biotin-NAD+ were added and the samples incubated for 30 min at 37 °C. Then, the proteins were subjected to SDS-PAGE, blotted onto a nitrocellulose membrane and the biotin-labelled, i.e., ADP-ribosylated, actin was detected by Western blotting with streptavidin-peroxidase and the ECL system. Intensity of biotin-actin was measured by densitometry using the Adobe Photoshop software (version 7.0, Adobe Systems GmbH, Munich, Germany, 2002).
3.6. Reproducibility of the Experiments and Statistics
All experiments were performed independently at least two times and results from representative experiments are shown in the figures. For quantification, the values (n = 3) were calculated as the means ± standard deviation (S.D.) with the Prism4 Software (GraphPad Software, Inc., La Jolla, CA, USA). Significance was tested with the Student t-test.
4. Conclusions
We have performed a series of experiments to demonstrate that the symmetrical positively charged β-cyclodextrin derivative, per-6-
S-(3-aminomethyl)benzylthio-β-cyclodextrin (AMBnTβ-CD), efficiently protects cultured epithelial cells from intoxication with the binary toxin CDT of
C. difficile. The more detailed investigation of the underlying mechanism strongly suggests that this compound inhibited the pH-dependent translocation of the enzyme component CDTa across cell membranes, which is mediated by trans-membrane pores formed by the separate binding/translocation component CDTb. This finding is in agreement with our recent data showing that AMBnTβ-CD blocks the translocation pores of the closely related binary C2 and iota toxins [
74,
76], thereby protecting cells from intoxication. This substance was originally generated as a tailored blocker for the translocation pore of the binary toxins of
Bacillus anthracis, protective antigen [
69], which shares the overall structure and mode of action with the translocation pores of the clostridial binary toxins [
22,
62]. Indirectly, our findings suggest that the CDTb pores, which have not been characterized as trans-membrane channels
in vitro so far, might play an essential role for the translocation of CDTa across membranes during uptake of CDT into the targeted mammalian cells.
However, the findings might also have an important medical implication since the observed inhibitory effects of AMBnTβ-CD suggest that this compound could serve as the broad-spectrum inhibitor against binary bacterial toxins that form oligomeric translocation channels to deliver their enzymatic active components into the host cell cytosol. Moreover, since the CDT-production contributes to the hypervirulence of C. difficile, AMBnTβ-CD might be an attractive lead compound to develop novel pharmacological strategies against these hypervirulent, CDT-producing strains.