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Human α-Defensins Inhibit Clostridium difficile Toxin B
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Human ␣-Defensins Inhibit Clostridium difficile Toxin B TORSTEN GIESEMANN, GREGOR GUTTENBERG, and KLAUS AKTORIES
See editorial on page 2174. Background & Aims: Clostridium difficile toxins A and B are major virulence factors implicated in pseudomembranous colitis and antibiotic-associated diarrhea. The toxins are glucosyltransferases, which inactivate Rho proteins involved in cellular signaling. Human ␣-defensins as part of the innate immune system inactivate various microbial pathogens as well as specific bacterial exotoxins. Here, we studied the effects of ␣-defensins human neutrophil protein (HNP)-1, HNP-3, and enteric human defensin (HD)-5 on the activity of C difficile toxins A and B. Methods: Inactivation of C difficile toxins by ␣-defensins in vivo was monitored by microscopy, determination of the transepithelial resistance of CaCo-2 cell monolayers, and analysis of the glucosylation of Rac1 in toxin-treated cells. In vitro glucosylation was used to determine Km and median inhibitory concentration (IC50) values. Formation of defensin-toxin complexes was analyzed by precipitation and turbidity studies. Results: Treatment of cells with human ␣-defensins caused loss of cytotoxicity of toxin B, but not of toxin A. Only ␣-defensins, but not -defensin-1 or cathelicidin LL-37, inhibited toxin B– catalyzed in vitro glucosylation of Rho guanosine triphosphatases in a competitive manner, increasing Km values for uridine 5=-diphosphate-glucose up to 10-fold. The IC50 values for inhibition of toxin B– catalyzed glucosylation by the ␣-defensins were 0.6 –1.5 mol/L. At high concentrations, defensins (HNP-1 >2 mol/L) caused high-molecular-mass aggregates, comparable to Bacillus anthracis protective antigen and lethal factor. Conclusions: Our data indicate that toxin B interacts with high affinity with ␣-defensins and suggest that defensins may provide a defense mechanism against some types of clostridial glucosylating cytotoxins.
C
lostridium difficile is a major cause of antibioticassociated diarrhea and pseudomembranous colitis.1–3 The main pathogenicity factors implicated in these diseases are the exotoxins toxin A and toxin B produced by the pathogen. After uptake, autocatalytic
processing, and translocation into target cells, the toxins mono-O-glucosylate and thereby inactivate lowmolecular-mass guanosine triphosphate– binding proteins of the Rho family. This leads to cytotoxic effects, including depolymerization of the actin cytoskeleton.4 – 6 In addition, infections with C difficile and exposure of colonic cells to the toxins initiate massive cellular responses, including neutrophil infiltration linked to the onset of inflammation with up-regulation and/or release of cytokines such as interleukin-8,7 interleukin-6,8 interleukin-1,9 leukotriene B4,10 and interferon gamma.11 The 2 major groups of human antimicrobial peptides, defensins and cathelicidins, play an important role in the innate immune response. They seem to be implicated in various gastrointestinal diseases (for review, see Wehkamp et al12,13). Human defensins are cationic, amphipathic peptides of 3.5– 6 kilodaltons that are characterized by 3 intramolecular disulfide bonds. They are subdivided into ␣- and -defensins. Until now, 4 ␣-defensins were isolated from neutrophils (human neutrophil protein [HNP]-1 to HNP-4). Additionally, 2 enteric human ␣-defensins (HD-5 and HD-6) are found in the granules of Paneth cells of the small intestine (for review, see De and Contreras14). The microbicidal potential of ␣-defensins is mainly based on the insertion of the amphipathic peptides into bacterial membranes and damage of membrane integrity.15 Next to this bactericidal action, the peptides have signaling effects and influence inflammation, proliferation, wound healing, release of cytokines, and chemotaxis (for review, see Kim and Kaufmann16 and Shi17). Recently, HNPs were described to neutralize bacterial exotoxins. Kim et al reported that HNP-1–3 inhibit lethal factor (LF) from Bacillus anthracis. HNP-1 inhibits the protease activity of the toxin, and injection of HNP-1–3 protected mice from the intoxication by LF.18 The same group showed that HNP-1 neutralizes mono-ADP-ribosylating toxins (eg, Pseudomonas aeruginosa exotoxin A and diphtheria toxin) in vitro and in vivo.19 Abbreviations used in this paper: GTPase, guanosine triphosphatase; hBD, human -defensin; HD, human defensin; HNP, human neutrophil protein; IC50, median inhibitory concentration; LF, lethal factor; LT, lethal toxin; SDS-PAGE, sodium dodecyl sulfate/polyacrylamide gel electrophoresis; TER, transepithelial resistance. © 2008 by the AGA Institute 0016-5085/08/$34.00 doi:10.1053/j.gastro.2008.03.008
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Institut für Experimentelle and Klinische Pharmakologie und Toxikologie, Universität Freiburg, Freiburg, Germany
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These findings with exotoxins prompted us to test whether ␣-defensins also neutralize clostridial glucosylating toxins. Here, we present evidence that ␣-defensins inhibit the catalytic activity and cytotoxicity of the most potent cytotoxic factor of C difficile, toxin B.
Materials and Methods Materials, Antibodies, and Proteins
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Recombinant human ␣-defensin HNP-1, HNP-3, enteric ␣-defensin HD-5, and human -defensin (hBD)-1 were from Peptides International, Inc (Louisville, KY). A mix of human native HNP-1–3 and cathelicidin LL-37 were from Panatecs GmbH (Tübingen, Germany). Uridine 5=-diphosphate (UDP)-[14C]glucose (287.4 mCi/ mmol) was from PerkinElmer (Rodau-Jügesheim, Germany). All chemicals used in this study were of analytical grade and purchased from commercial sources. Anti-Rac1 monoclonal antibody clone 23A8, reacting with glucosylated and nonglucosylated Rac1, was from Upstate (Millipore Corp, Schwalbach, Germany); antiRac1 monoclonal antibody clone 102 recognizing only nonglucosylated Rac1 was from BD Biosciences Pharmingen (Heidelberg, Germany). Native toxins A and B from C difficile VPI 10463 and native lethal toxin (LT) from Clostridium sordellii 6018 were purified as described.20 Toxin A was additionally purified by thyroglobulin affinity chromatography.21 The catalytic domain of toxin B (amino acids 1–546) was expressed as glutathione S-transferase fusion proteins from the Escherichia coli expression vector pGEX-2T (Amersham Biosciences, Freiburg, Germany) as reported previously.22 The recombinant guanosine triphosphate– binding proteins RhoA, Rac1, and Cdc42 were prepared as glutathione S-transferase fusion proteins as described.23 The Bacillus anthracis toxins LF and protective antigen were a kind gift from R. J. Collier (Boston Medical School, Boston, MA).
Cell Culture and In Vivo Intoxication Experiments Swiss 3T3 and CaCo-2 cells were cultivated in Dulbecco’s modified Eagle medium at 37°C and 5% CO2 with medium containing 10% heat-inactivated fetal calf serum, 2 mmol/L L-glutamate, 100 U/mL penicillin, and 100 g/mL streptomycin and were trypsinized and reseeded 2 or 3 times a week. For all in vivo intoxication assays, the corresponding cells were washed twice with and kept in fetal calf serum–free Dulbecco’s modified Eagle medium. Antimicrobial peptides (1 and/or 3 mol/L, as indicated) were applied to the cells before toxin A or toxin B (5–100 pmol/L) was added. Untreated cells, either defensin alone or toxin A and toxin B alone, served as controls, respectively. For microscopic studies, Swiss 3T3 cells were grown in 24-well dishes and intoxicated with 50 pmol/L toxin A or 5 and 50 pmol/L toxin B after pretreatment with de-
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fensins (5 minutes or 3 hours, respectively). Pictures were taken with an AxioCam HRC camera and Axio Vision software (Carl Zeiss AG, Oberkochen, Germany). For the transepithelial resistance (TER) assay, CaCo-2 cells were seeded on 12-well Millicell cell culture inserts (Millipore Corp) and incubated 4 – 6 days with medium exchange every 2 days. Assays were performed when TER values reached ⬃1000 to 2000 ⍀/cm2. TER was determined with Endohm-12 (World Precision Instruments, Sarasota, FL). Defensins were preincubated with the cells for 2.5 hours before 100 pmol/L toxin A or toxin B was applied to the upper compartment of the Millicell cell culture inserts, and decrease of TER was monitored for up to 8 hours. For the detection of the glucosylation of Rac1 after preincubation with defensins (for 2.5 hours) and intoxication with 50 pmol/L holotoxin B, Swiss 3T3 cells grown in 24-well dishes were used. Sixty minutes after toxin application, the cells were washed with phosphate-buffered saline and lysed with Laemmli buffer, followed by Western blotting. Rac1 was detected using monoclonal antibodies clone 23A8 (1:5000) and clone 102 (1:10,000), followed by an anti-mouse immunoglobulin G horseradish peroxidase secondary antibody (1:5000; Biotrend, Cologne, Germany). Corresponding bands were detected using enhanced chemoluminescence.
In Vitro Glucosylation Experiments For initial studies, 9 nmol/L toxin B and 3 mol/L Rac1 were incubated at 37°C in the presence of 10 mol/L UDP-[14C]glucose in IVG buffer containing 50 mmol/L HEPES (pH 7.5), 100 mmol/L KCl, 2 mmol/L MgCl2, and 1 mmol/L MnCl2. After 2 minutes, defensins were added to a final concentration of 1 or 3 mol/L. Samples of 20 L were taken at indicated time points. Km values were determined with 1 nmol/L toxin B1–546, 1 mol/L Rac1, defensins HNP-1 and HNP-3 (final concentration, 1 mol/L), or HD-5 (3 mol/L) and varying UDP-[14C]glucose concentrations (0.5–20 mol/L). The reactions were incubated for 10 minutes at 37°C in IVG buffer. For determination of the median inhibitory concentration (IC50) values, 3 nmol/L toxin B or toxin B1–546 were preincubated with HNP-1 or HD-5 (0.1–5 mol/L) for 10 minutes at 37°C in the presence of 10 mol/L UDP[14C]glucose in IVG buffer supplemented with 100 g/mL bovine serum albumin. Glucosylation reaction was started by addition of 1 mol/L recombinant Rac1, RhoA, or Cdc42, respectively, and incubated for 15 minutes at 37°C. Total volume was 20 L. End point inhibition and reversibility of inhibition were examined with 3 nmol/L toxin B1–546, 1 mol/L recombinant or native defensins, and 5.5 mol/L Rac1. Toxin and antimicrobial peptides were preincubated for 15 minutes at 37°C in IVG buffer in a total volume of 22.5 L. The solution was split into 2 fractions (20 and 2.5 L) and the volume adjusted to 25 L each by
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addition of UDP-[14C]glucose (10 mol/L) and Rac1 (5.5 mol/L). Reactions were incubated for 10, 30, or 100 minutes at 37°C. UDP-[14C]glucose–labeled proteins were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) followed by phosphoimaging and densitometry. Quantification of signals and determination of IC50 and Km values were performed with ImageQuant (Amersham Biosciences) and SigmaPlot/Enzyme Kinetics module (Systat Software, Erkrath, Germany), respectively.
UDP-Sugar Hydrolase Assay
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UDP-sugar hydrolysis was essentially performed as described.23 Toxin B1–546 (100 nmol/L) pretreated with HNP-1 or HD-5 (0 –10 mol/L; 10 minutes, 37°C) was incubated with 20 mol/L UDP-[14C]glucose and 60 mol/L unlabeled UDP-glucose in a buffer containing 50 mmol/L HEPES (pH 7.5), 100 mmol/L KCl, 2 mmol/L MgCl2, 100 mol/L bovine serum albumin, and 100 mol/L MnCl2 for 30 minutes at 30°C. Samples were subjected to thin-layer chromatography with polyethyleneimine-cellulose plates (Merck, Darmstadt, Germany) and 0.2 mmol/L LiCl to separate the hydrolyzed sugar from UDP-sugars. The plates were dried and analyzed by Phosphorimager analysis (GE Healthcare, München, Germany). Quantification was performed with ImageQuant.
Aggregation Studies For turbidity assays, 3 g of each toxin (as indicated) were diluted in 500 L IVG buffer and incubated for 100 seconds before varying concentrations (0.5–3 mol/L) of the indicated peptides were injected in a volume of 50 L IVG buffer. Formation of aggregates was monitored by measurement of the increasing absorbance (light scattering) at 600 nm over time (up to 20 minutes) with a PerkinElmer LS 50 B luminescence spectrometer. Precipitation studies were performed either with 1.1 g toxin B1–546 or 2–3 g toxin B, B anthracis LF, B anthracis protective antigen, and C sordellii LT in a total volume of 25 L. Before addition of defensins, the protein solutions were centrifuged for 15 minutes at 13,000g and 4°C to segregate possible preformed protein complexes. The supernatants were incubated at rising concentrations of HNP-1 or HD-5 (0 –20 mol/L) for 10 minutes at 37°C. Following centrifugation (4°C, 45 minutes, 13,000g), supernatants and pellets were separated and transferred to SDS-PAGE. Proteins were visualized by Coomassie blue staining of the gels.
Results Cytotoxicity of Toxin B, but Not of Toxin A, Is Diminished by ␣-Defensins To test whether defensins and/or cathelicidin protect cells against the cytotoxic effects of clostridial glu-
Figure 1. ␣-Defensins neutralize toxin B, but not toxin A, in cell culture. Swiss 3T3 cells were preincubated with 3 mol/L HNP-1 or HD-5 in fetal calf serum–free medium. After 3 hours, the cells were intoxicated with 50 pmol/L toxin B or toxin A as indicated. Cell rounding was monitored by light microscopy and pictures were taken after 1 hour (toxin B) or 5 hours (toxin A). Corresponding controls as indicated.
cosylating toxins, we applied a mix of native HNP-1–3, recombinant HNP-1, HNP-3, HD-5, hBD-1, and cathelicidin LL-37 (3 mol/L each) to Swiss 3T3 cells before toxin application. The intoxication process characterized by cell rounding was monitored by light microscopy (exemplarily shown for HNP-1 and HD-5 in Figure 1). For toxin B, but not for toxin A, we found a strong reduction in cytotoxicity when the cells were pretreated with 3 mol/L of the corresponding ␣-defensins. In contrast, neither hBD-1 nor LL-37 protected cells from toxin B–induced cell rounding (not shown). HNPs were more effective than HD-5, which conferred only partial protection at a concentration of 3 mol/L. At 1 mol/L, the defensins did not protect or only weakly protected the cells against toxin B (not shown). The protective effect of HNP-1 persisted for up to 24 hours. Results were repro-
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duced with several cell lines (eg, HeLa, CaCo-2, HT-29, and Vero cells), excluding a cell type– dependent effect (not shown). To quantify reduction in cytotoxicity, the toxin-induced decrease in TER of human intestinal cells grown in a monolayer (CaCo-2 cells) was measured. As shown in Figure 2A, toxin B treatment from the apical site of the
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cell monolayer leads to a fast and strong reduction of the TER, with complete intoxication reached after about 4 –5 hours. HNP-1 3 mol/L strongly delayed intoxication with toxin B. HNP-1 1 mol/L displayed a weak protective effect (Figure 2A). Again, HD-5 was less effective even at a final concentration of 3 mol/L (Figure 2B). For toxin A, we found no significant influence of either defensin on the cytotoxic potential (Figure 2C), corroborating the microscopic studies. An Rac1 antibody that recognizes only nonglucosylated Rac124,25 was used to monitor the glucosylation status of Rac1 after intoxication of cells pretreated with the corresponding defensins (as described previously). HNP-1 and HD-5 inhibited toxin B–induced glucosylation of Rac1 in a concentration-dependent manner (Figure 2D). Again, HNP-1 displayed a stronger effect than HD-5 with complete inhibition of glucosylation at a final concentration of 3 mol/L. The same concentration of HD-5 reduced the glucosylation only to about 30%.
␣-Defensins Inhibit Toxin B–Mediated Glucosylation of Small Guanosine Triphosphatases
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The inhibiting effects of ␣-defensins on glucosyltransferase activity of toxin B were studied in an in vitro glucosylation assay. First, reactions with toxin A and toxin B were started without defensins. HNP-1, HNP-3, or HD-5 (1 and 3 mol/L each) were added after 2 minutes to the reaction mixture. In case of toxin B, HNP-1 (Figure 3A) and HNP-3 (Figure 3B) led to an immediate and concentrationdependent decrease of the glucosyltransferase activity with strong or even complete inhibition at 3 mol/L. Inhibition by HD-5 was delayed with an onset of a decline after 5 minutes (at 3 mol/L) or 10 minutes (at 1 mol/L, Figure 3C), respectively. A mix of native HNPs (HNP-1–3) exhibited strong inhibition of glucosyltransferase activity comparable to recombinant HNP-1 and HNP-3 (Supplementary Figure 1A; see supplementary material online at www.gastrojournal. org). Glucosyltransferase activity of toxin A was not influenced by addition of HNP-1 (Supplementary Figure 1B; see supplementary material online at www.gastrojournal.org) or a mix of native HNP-1–3 (not shown).
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Figure 2. ␣-Defensins delay toxin B–mediated intoxication in vivo by inhibition of glucosylation of small GTPases. (A–C) Human intestinal epithelial cells (CaCo-2) grown on Millicell filter inserts were preincubated with 1 and 3 mol/L HNP-1 or HD-5 for 2.5 hours before toxin A or toxin B (100 pmol/L each) was added (as indicated). Decrease of TER was monitored at indicated time points. Starting resistance was set to 100%, and TER values are given as percent of starting resistance. “Control,” untreated cells. (D) Swiss 3T3 cells were preincubated with indicated ␣-defensins and intoxicated with 50 pmol/L toxin B as described previously. After onset of cell rounding (60 minutes), cells were lysed and transferred to SDS-PAGE and Western blotting. Rac1 was detected with 2 different monoclonal anti-Rac1 antibodies, one recognizing unmodified and glucosylated Rac1 (“total Rac1,” input control) and the second recognizing only unmodified Rac1 (“non-Glc Rac1”). Controls without toxin as indicated.
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The corresponding Km values for HNP-1, HNP-3, and HD-5 were determined using the catalytic domain of toxin B encompassing amino acids 1–546 (toxin B1–546), Rac1, and varying UDP-[14C]glucose concentrations (0.5–20 mol/L). Defensins and UDP-glucose were added simultaneously to the reaction. Control glucosylation reaction without defensin resulted in a Km value of 2.4 mol/L. Addition of ␣-defensins inhibited glucosyltransferase reaction in a competitive manner, with HNP-1 and HNP-3 elevating corresponding Km values about 10-fold to 23.8 mol/L (HNP-1, Figure 3D) and about 9-fold to 21.1 mol/L (HNP-3, Figure 3E), respectively. At 1 mol/L, HD-5 elevated Km only by the factor 2 (not shown). When the concentration of HD-5 was raised to 3 mol/L, the Km value increased about 6- to 7-fold (Figure 3F).
Reversibility of End Point Inhibition Next to recombinant and native human ␣-defensins, hBD-1 and cathelicidin LL-37 were tested for their inhibitory potential. To guarantee maximal inhibition, the defensins and toxin B1–546 were preincubated for 15 minutes at 37°C before Rac1 (5.5 mol/L) and UDP-
BASIC– ALIMENTARY TRACT
Figure 3. Characterization of inhibition of toxin B by ␣-defensins in vitro. (A–C) Glucosylation reactions were started without defensins in the presence of 5.5 mol/L Rac1 and 9 nmol/L toxin B. (A) HNP-1, (B) HNP-3, or (C) HD-5 were added after 2 minutes in a final concentration of 1 or 3 mol/L, respectively. Samples were taken at the indicated time points, and the amount of [14C]-labeled Rac1 was analyzed by SDS-PAGE, autoradiography, and densitometry. Signal intensity of samples without defensin at t ⫽ 10 or 25 minutes was set to 100%, and relative glucosyltransferase activity of each sample is shown as percent glucosylation of the corresponding control (n ⱖ 3 ⫾ SEM). (D–F) Km values of samples without defensin (“control”), with (D) 1 mol/L HNP-1, (E) 1 mol/L HNP-3, or (F) 3 mol/L HD-5 were determined at varying UDP-[14C]glucose concentrations (0.5–20 mol/L) in the presence of 1 mol/L Rac1 and 1 nmol/L toxin B1–546. Corresponding Michaelis–Menten kinetics (n ⱖ 3 ⫾ SD) and respective Km values are shown. Glucosyltransferase activity (rate [1/s]) of toxin B1–546 is given as pmol glucosylated Rac1⫺1 · pmol enzyme⫺1 · s.
[14C]glucose (10 mol/L) were added. These conditions were found to enhance defensin-mediated inhibition (see Supplementary Figure 2; see supplementary material online at www.gastrojournal.org). Neither hBD-1 nor LL-37 reduced glucosyltransferase activity of toxin B (Figure 4A), corroborating the in vivo data from cell intoxication. Reversibility of defensin-induced inhibition was assessed by diluting the preincubated defensin/toxin mix by a factor of 10, resulting in a final concentration of 0.1 mol/L defensin and 0.3 nmol/L toxin B1–546 at constant Rac1 and UDP-[14C]glucose concentrations. This dilution partially reconstituted glucosyltransferase activity, indicating reversibility of defensin-mediated inhibition. To visualize corresponding signals in a comparable manner, undiluted samples were incubated for 10 minutes and diluted samples for 100 minutes (Figure 4A). For statistical analysis (Figure 4B), all probes were incubated for 30 minutes and signals normalized against corresponding controls. Partial reversibility of the inhibiting effects suggested that the overall structure of the toxin was not changed to a large extent by the defensins. This view
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was supported by the finding that the sensitivity of the toxin toward V8-protease was not strongly affected by the presence of HNPs (Supplementary Figure 3; see supplementary material online at www.gastrojournal .org). Partial protection of cleavage observed with HD-5 may be based on the strong aggregation of toxin B (see following text) and reduced accessibility of cleavage sites. To determine whether the 3-dimensional structure of defensins is crucial for their inhibitory potential, HNP-1 and HD-5 were incubated with the reducing agent dithiothreitol or heated at 95°C for 5 minutes. The corresponding defensins were then applied in the in vitro glucosylation assay as described previously. Treatment with dithiothreitol (10 mmol/L) largely abolished the inhibitory potential of HNP-1 and HD-5 (Figure 4C), indicating that an intact overall structure stabilized by internal disulfide bonds is essential for the inhibition. Heating of the peptides to 95°C did not influence the inhibitory potential.
Determination of IC50 Values
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Figure 4. End point inhibition: specificity, reversibility, and sensitivity toward dithiothreitol. (A and B) To guarantee maximal inhibition by the antimicrobial peptides, toxin B1–546 (3 nmol/L) was preincubated for 15 minutes at 37°C with indicated defensins or cathelicidin LL-37 (1 mol/L each). Part of the sample was diluted 1:10 before Rac1 (5.5 mol/L) and UDP[14C]glucose (10 mol/L) were added to start glucosylation. Samples were incubated for 10 minutes (“undiluted”), 100 minutes (“diluted”), or 30 minutes (B). Radioactive labeled Rac1 was detected by SDS-PAGE and autoradiography. (A) Reappearing signals indicate partial reversibility of the inhibition. (B) Statistical analysis of signal intensities normalized against corresponding controls (n ⫽ 3 ⫾ SEM). (C) Toxin B1–546 (3 nmol/L) was preincubated with HNP-1 or HD-5 (3 mol/L each) ⫾ 10 mmol/L dithiothreitol in a total volume of 20 L for 10 minutes at 37°C. Heating of defensins (95°C, 5 minutes) was performed before preincubation with the toxin. Toxin B1–546 pretreated only with reducing agents served as control. Glucosylation reaction was initiated by addition of 1 mol/L Rac1. Samples were taken after 15 minutes, and radioactively labeled Rac1 was detected by SDS-PAGE and autoradiography.
The concentration-dependent effect of HNP-1 and HD-5 on the glucosylation of the main substrates of toxin B, namely RhoA, Rac1, and Cdc42 (1 mol/L each), was tested by the in vitro glucosylation assay. To guarantee maximal inhibition, a preincubation step of 10 minutes (as described previously) was applied. In all cases, treatment of the toxins with either HNP-1 or HD-5 strongly reduced the glucosylation of the low-molecularmass guanosine triphosphatases (GTPases) with IC50 values ranging from 0.6 to 1.5 mol/L, estimated from the corresponding regression curves (exemplarily represented in Figure 5A and summarized in Table 1). As expected, HNP-1 displayed slightly lower IC50 values compared with HD-5. Based on the preincubation step, which counteracts the delayed onset of inhibition by HD-5 (see Figure 3C), the differences between HNP-1 and HD-5 are not as pronounced as seen for the corresponding Km values. Next to the inhibition of glucosyltransferase activity, ␣-defensins also inhibited glucohydrolase activity of toxin B1–546 (HNP-1: IC50 ⫽ 3.8 mol/L; HD-5: IC50 ⫽ 6.8 mol/L; see Figure 5B), corroborating a direct effect of the defensins on the catalytic activity of the toxin.
Toxin Aggregation Induced by Defensins Under various experimental settings utilizing high defensin concentrations (eg, for gel filtration analysis), we observed that defensins induced aggregation of toxin B. Apparently these aggregates were of high molecular mass because they were precipitated at low g-force (13,000g). To determine the concentrations of ␣-defensin, which caused complex formation and/or precipitation of toxin B, we performed a precipitation
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assay after preincubation of toxin B with rising concentrations of HNP-1. In case of toxin B1–546, which proved to be most sensitive to formation of aggregates (Supplementary Figure 4; see supplementary material online at www.gastrojournal.org), precipitation started at ⬃2 mol/L HNP-1 (Figure 6A). As shown in Figure 6A, HNP-1 was shifted to the pellet fraction together with the toxin. Estimated from Coomassie blue– stained gels, approximately 18 pmol toxin B1–546 coprecipitated up to ⬃250 pmol HNP-1, indicating multiple binding sites for HNP-1. Comparable precipitation studies were performed with toxin A, toxin B, LT, and B anthracis LF and protective antigen in combination with HNP-1 and HD-5 (Supplementary Figure 4; see supplementary material online at www.gastrojournal .org). Except LT and toxin A, which were not inhibited by the corresponding defensins, all toxins tested formed aggregates. HD-5 precipitated toxins more efficiently than HNP-1. Aggregate formation was further characterized by turbidity assays. Again, HD-5 proved to be most potent in aggregate formation in combination with toxin B1–546, followed by HNP-1 and HNP-3 (Figure 6B). Neither hBD-1 nor cathelicidin LL-37 induced any detectable aggregation, excluding a mere charge effect. Although full-length toxin B is much larger in size than the isolated catalytic domain, it proved to aggregate to the same extent or even less. Accordingly, aggregation was mainly mediated by the N-terminal part of toxin B (amino acids 1–546). B anthracis LF and protective antigen also formed aggregates under the same conditions (Supplementary Figure 5; see supplementary material online at www.gastrojournal.org).
Discussion Here we report that the ␣-defensins HNP-1, HNP-3, and enteric HD-5 prevent the cytotoxic effects of
Table 1. IC50 Values Figure 5. Determination of the IC50 values for glucosyltransferase and glucohydrolase activity. (A) Glucosyltransferase activity. Toxin B (3 nmol/L) was preincubated with rising concentrations (0.1–5 mol/L) of HNP-1 or HD-5 in a total volume of 20 L for 10 minutes at 37°C. Glucosylation reaction was started by adding 1 mol/L Rac1. Radioactively labeled Rac1 was detected by SDS-PAGE and autoradiography followed by densitometry for statistical evaluation. Total Rac1 (input control) is shown by Coomassie blue–stained gels (upper panels, as indicated). (B) Glucohydrolase activity. Toxin B1–546 (100 nmol/L) was preincubated with rising concentrations (0 –10 mol/L) of HNP-1 or HD-5 in a total volume of 10 L for 10 minutes at 37°C. Glucohydrolase reaction was started by the addition of 20 mol/L UDP-[14C]glucose. Incubation was for 30 minutes at 30°C. UDP-[14C]glucose hydrolyzed was analyzed by thin-layer chromatography, autoradiography, and densitometry. (A and B) Signal intensity of samples without defensin was set to 1.0, and relative glucosyltransferase/glucohydrolase activity of each sample is shown (n ⱖ 3 ⫾ SD). IC50 values for the main substrates of toxin B were determined from the corresponding regression curves (see Table 1).
Toxin (3 nmol/L) Toxin B
GTPase (1 mol/L) Rac1 RhoA Cdc42
Toxin B1–546
Rac1 RhoA Cdc42
Defensin (0–5 mol/L)
IC50 value (mol/L)
HNP-1 HD-5 HNP-1 HD-5 HNP-1 HD-5 HNP-1 HD-5 HNP-1 HD-5 HNP-1 HD-5
0.6 0.8 0.8 1.1 0.7 0.6 0.6 1.2 1.0 1.5 0.7 1.5
NOTE. Listed are all determined combinations of toxin B and toxin B1–546 with the main protein substrates Rac1, RhoA, and Cdc42, respectively. IC50 values were determined as described in Figure 5.
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Figure 6. Toxin aggregation induced by ␣-defensins. (A) Toxin B1–546 was preincubated for 15 minutes at 37°C with rising concentrations of HNP-1 (0 –20 mol/L). Samples were centrifuged at 13,000g at 4°C for 50 minutes. Supernatants were separated from pellets and the individual fractions transferred to SDS-PAGE. Coomassie blue–stained gels exhibiting the toxin protein (63 kilodaltons) and defensin (3.5 kilodaltons) bands are shown. (B) A solution containing 3 g toxin B1–546 was incubated for 100 seconds before 3 mol/L (final concentration) of the indicated antimicrobial peptides was injected. Aggregate formation in solution was monitored by increase in turbidity at OD600 over time. Intensity (Int) of light scattering is given as arbitrary units.
toxin B in intestinal epithelial cells and in a large array of other cell types. The ␣-defensins inhibited the glucosylation of Rho proteins by toxin B, which is suggested to be the molecular basis for the cytotoxic effects of the toxin. Because the defensins blocked the glucohydrolase activity of toxin B and did not affect the GTPase activities of Rho proteins per se (not shown), our data indicate that the inhibiting effect is caused by blockade of the catalytic activity of the toxin. This view is supported by the finding
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that ␣-defensins compete with the binding of UDP-glucose to the glucosyltransferase domain of toxin B. The substrate competition at the active site is characterized by an up to 10-fold increase in the Km value for UDPglucose. The inhibition is specific for toxin B, because toxin A is not influenced by the defensins under the same conditions. The effect is specific for ␣-defensins, because hBD-1 and cathelicidin LL-37 did not influence glucosyltransferase activity. The dilution experiments suggest that inhibition of glucosyltransferase activity by defensins is reversible at least at low concentrations of the inhibitors, which is in agreement with the Michaelis– Menten kinetics obtained. The unaltered sensitivity of HNP-treated toxin B toward proteolytic cleavage by V8protease (Supplementary Figure 3; see supplementary material online at www.gastrojournal.org) strongly suggests that the binding of the ␣-defensins to toxin B does not cause major changes in the toxin structure and does not merely result in a denaturating effect. Regarding the concentration-dependent inhibition, we obtained IC50 values at a low range (0.6 –1.5 mol/L) for toxin B and HNP-1 or HD-5. Defensins occur at very high concentrations in specific intestinal parts. Recently, it has been estimated that 0.5 mg of HD-5 is stored per square centimeter of ileal mucosa with concentrations of 50 – 250 g/mL (⬃14 to 70 mol/L) in the intestinal lumen.26 Thus, this concentration would largely block the action of toxin B, suggesting a protective role of defensins toward the potent effects of C difficile toxin B. HD-5 is produced by Paneth cells, which are usually in the small intestine but not in the colon, and only metaplastic Paneth cells are observed in the inflamed colon. However, C difficile is also capable of colonizing the small intestine, where it may act as an asymptomatic reservoir for disease.27 Whether the low virulence of C difficile in the small intestine is related to the occurrence of toxininactivating peptides remains to be clarified. Our findings add another exotoxin to the list of bacterial toxins, which were reported to be inhibited by defensins. Among these are the LF from B anthracis, diphtheria toxin, and Pseudomonas exotoxin A. Interestingly, B anthracis LF (IC50 ⫽ ⬃0.2 mol/L) and the mono-ADP-ribosyltransferases diphtheria toxin (IC50 ⫽ ⬃11.1 mol/L) and exotoxin A (IC50 ⫽ ⬃19.2 mol/L) are inhibited at similar concentrations of defensins as observed for toxin B.18,19 Also, the small concentration range observed for inhibition of toxin B with a weak protection at 1 mol/L and almost complete protection at 3 mol/L is in line with the action of HNP-1 on anthrax LT.18 Thus, the interaction of defensins with these toxins might be based on related mechanisms. The direct effect of defensins on catalytic activity is a plausible cause for their inhibiting potential. However, we cannot exclude additional effects on the receptor and/or uptake of toxin B in vivo. We observed a concentration-dependent aggregation of toxin B in the presence of ␣-defensins. Notably, the effi-
ciency of ␣-defensins to induce aggregation of toxin B is not paralleled by inhibition of glucosyltransferase activity. HD-5 induced formation of aggregates more efficiently than HNP-1, but it was less efficient than HNP-1 in inhibiting glucosyltransferase activity. Also, HNPs inactivated glucosyltransferase activity of holotoxin B immediately, whereas aggregation started with a significant delay. Regarding the in vivo situation, it is noteworthy that aggregation of holotoxin B by HNP-1 started at defensin concentrations higher than the corresponding IC50 values. Therefore, aggregate formation is, at least in part, independent of the inhibitory effects of HNPs. Neither toxin A nor LL-37 nor hBD-1 displayed aggregate formation. In the case of HD-5, we cannot rule out an influence of strong aggregate-forming potential on the observed inhibitory activity. The complex formation seems to be a common feature, because B anthracis LF and protective antigen, which were used as control proteins in this context, displayed the same behavior. Therefore, we believe that this observation may represent an additional mode of action for specific defensins (eg, HD-5) or at sites where high defensin concentrations are present, for example, within specialized regions of the small intestine or phagocytic vacuoles of neutrophils. We were surprised to find that in contrast to toxin B, toxin A was not significantly inactivated by HNP-1 under the in vivo conditions tested. The same was true for C sordellii LT, which is even more similar in structure to toxin B than toxin A. These different effects observed with toxin B and toxin A or LT indicate the specificity of the interaction of the defensins with toxin B. Early studies on C difficile infections and pathogenicity referred to “enterotoxin A” as the causative agent for pseudomembranous colitis and antibiotic-associated diarrhea.28 Based mainly on the frequent isolation of toxin A–negative, toxin B–positive strains from patients with C difficile–associated diseases, the potent “cytotoxin B” seems to be the responsible pathogenic agent in many cases.29 –32 In this respect, the selectivity of human ␣-defensins HNP-1, HNP-3, and HD-5 for toxin B is remarkable. It remains to be clarified whether a high-affinity interaction of toxin B with defensins is at least in part responsible for the former characterization of toxin B as a cytotoxin without enterotoxic activity, which was deduced from various experimental animal colitis models. Taken together, we observed that defensins interact with high affinity with C difficile toxin B to inhibit its activity. This finding offers a new perspective in host-pathogen interaction in C difficile infections and associated diseases.
Supplementary Data Note: To access the supplementary material accompanying this article, visit the online version of Gastroenterology at www.gastrojournal.org, and at doi:10.1053/j.gastro. 2008.03.008.
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References 1. Bartlett JG, Perl TM. The new Clostridium difficile—what does it mean? N Engl J Med 2005;353:2503–2505. 2. Bartlett JG, Moon N, Chang TW, et al. Role of Clostridium difficile in antibiotic-associated pseudomembranous colitis. Gastroenterology 1978;75:778 –782. 3. Kelly CP, LaMont JT. Clostridium difficile infection. Annu Rev Med 1998;49:375–390. 4. Voth DE, Ballard JD. Clostridium difficile toxins: mechanism of action and role in disease. Clin Microbiol Rev 2005;18:247–263. 5. Just I, Gerhard R. Large clostridial cytotoxins. Rev Physiol Biochem Pharmacol 2004;152:23– 47. 6. Thelestam M, Chaves-Olarte E. Cytotoxic effects of the Clostridium difficile toxins. Curr Top Microbiol Immunol 2000;250:85–96. 7. Meyer GK, Neetz A, Brandes G, et al. Clostridium difficile toxins A and B directly stimulate human mast cells. Infect Immun 2007; 75:3868 –3876. 8. Ng EK, Panesar N, Longo WE, et al. Human intestinal epithelial and smooth muscle cells are potent producers of IL-6. Mediators Inflamm 2003;12:3– 8. 9. Tixier E, Lalanne F, Just I, et al. Human mucosa/submucosa interactions during intestinal inflammation: involvement of the enteric nervous system in interleukin-8 secretion. Cell Microbiol 2005;7:1798 –1810. 10. McVey DC, Vigna SR. The role of leukotriene B4 in Clostridium difficile toxin A-induced ileitis in rats. Gastroenterology 2005; 128:1306 –1316. 11. Ishida Y, Maegawa T, Kondo T, et al. Essential involvement of IFN-gamma in Clostridium difficile toxin A-induced enteritis. J Immunol 2004;172:3018 –3025. 12. Wehkamp J, Schauber J, Stange EF. Defensins and cathelicidins in gastrointestinal infections. Curr Opin Gastroenterol 2007;23:32–38. 13. Wehkamp J, Fellermann K, Herrlinger KR, et al. Mechanisms of disease: defensins in gastrointestinal diseases. Nat Clin Pract Gastroenterol Hepatol 2005;2:406 – 415. 14. De SK, Contreras R. Human antimicrobial peptides: defensins, cathelicidins and histatins. Biotechnol Lett 2005;27:1337–1347. 15. Zasloff M. Antimicrobial peptides of multicellular organisms. Nature 2002;415:389 –395. 16. Kim C, Kaufmann SH. Defensin: a multifunctional molecule lives up to its versatile name. Trends Microbiol 2006;14:428 – 431. 17. Shi J. Defensins and Paneth cells in inflammatory bowel disease. Inflamm Bowel Dis 2007;13:1284 –1292. 18. Kim C, Gajendran N, Mittrucker HW, et al. Human alpha-defensins neutralize anthrax lethal toxin and protect against its fatal consequences. Proc Natl Acad Sci U S A 2005;102:4830 – 4835. 19. Kim C, Slavinskaya Z, Merrill AR, et al. Human alpha-defensins neutralize toxins of the mono-ADP-ribosyltransferase family. Biochem J 2006;399:225–229. 20. Just I, Selzer J, Hofmann F, Aktories K. Clostridium difficile toxin B as a probe for Rho GTPases. In: Aktories K, ed. Bacterial toxins — tools in cell biology and pharmacology. Weinheim: Chapman & Hall, 1997:159 –168. 21. Krivan HC, Wilkins TD. Purification of Clostridium difficile toxin A by affinity chromatography on immobilized thyroglobulin. Infect Immun 1987;55:1873–1877. 22. Hofmann F, Busch C, Prepens U, et al. Localization of the glucosyltransferase activity of Clostridium difficile toxin B to the N-terminal part of the holotoxin. J Biol Chem 1997;272:11074 –11078. 23. Just I, Wilm M, Selzer J, et al. The enterotoxin from Clostridium difficile (ToxA) monoglucosylates the Rho proteins. J Biol Chem 1995;270:13932–13936. 24. Genth H, Huelsenbeck J, Hartmann B, et al. Cellular stability of Rho-GTPases glucosylated by Clostridium difficile toxin B. FEBS Lett 2006;580:3565–3569.
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25. Egerer M, Giesemann T, Jank T, et al. Auto-catalytic cleavage of Clostridium difficile toxins A and B depends on a cysteine protease activity. J Biol Chem 2007;282:25314 –25321. 26. Ghosh D, Porter E, Shen B, et al. Paneth cell trypsin is the processing enzyme for human defensin-5. Nat Immunol 2002;3: 583–590. 27. Testore GP, Nardi F, Babudieri S, et al. Isolation of Clostridium difficile from human jejunum: identification of a reservoir for disease? J Clin Pathol 1986;39:861– 862. 28. Lyerly DM, Saum KE, MacDonald DK, et al. Effects of Clostridium difficile toxins given intragastrically to animals. Infect Immun 1985;47:349 –352. 29. Komatsu M, Kato H, Aihara M, et al. High frequency of antibiotic associated diarrhea due to toxin A-negative, toxin B-positive Clostridium difficile in a hospital in Japan and risk factors for infection. Eur J Clin Microbiol Infect Dis 2003;22:525–529. 30. Savidge TC, Pan W-H, Newman P, et al. Clostridium difficile toxin B is an inflammatory enterotoxin in human intestine. Gastroenterology 2003;125:413– 420.
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31. van den Berg RJ, Claas EC, Oyib DH, et al. Characterization of toxin A-negative, toxin B-positive Clostridium difficile isolates from outbreaks in different countries by amplified fragment length polymorphism and PCR ribotyping. J Clin Microbiol 2004; 42:1035–1041. 32. Samra Z, Talmor S, Bahar J. High prevalence of toxin A-negative toxin B-positive Clostridium difficile in hospitalized patients with gastrointestinal disease. Diagn Microbiol Infect Dis 2002;43: 189 –192.
Received October 16, 2007. Accepted March 6, 2008. Address requests for reprints to: Klaus Aktories, Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Albertstrasse 25, D-79104 Freiburg, Germany. e-mail: klaus.aktories@pharmakol. uni-freiburg.de; fax: (49) 761-2035311. Supported by the Deutsche Forschungsgemeinschaft. The authors report that no conflicts of interest exist.
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Human ␣-Defensins Inhibit Clostridium difficile Toxin B TORSTEN GIESEMANN, GREGOR GUTTENBERG, and KLAUS AKTORIES
See editorial on page 2174. Background & Aims: Clostridium difficile toxins A and B are major virulence factors implicated in pseudomembranous colitis and antibiotic-associated diarrhea. The toxins are glucosyltransferases, which inactivate Rho proteins involved in cellular signaling. Human ␣-defensins as part of the innate immune system inactivate various microbial pathogens as well as specific bacterial exotoxins. Here, we studied the effects of ␣-defensins human neutrophil protein (HNP)-1, HNP-3, and enteric human defensin (HD)-5 on the activity of C difficile toxins A and B. Methods: Inactivation of C difficile toxins by ␣-defensins in vivo was monitored by microscopy, determination of the transepithelial resistance of CaCo-2 cell monolayers, and analysis of the glucosylation of Rac1 in toxin-treated cells. In vitro glucosylation was used to determine Km and median inhibitory concentration (IC50) values. Formation of defensin-toxin complexes was analyzed by precipitation and turbidity studies. Results: Treatment of cells with human ␣-defensins caused loss of cytotoxicity of toxin B, but not of toxin A. Only ␣-defensins, but not -defensin-1 or cathelicidin LL-37, inhibited toxin B– catalyzed in vitro glucosylation of Rho guanosine triphosphatases in a competitive manner, increasing Km values for uridine 5=-diphosphate-glucose up to 10-fold. The IC50 values for inhibition of toxin B– catalyzed glucosylation by the ␣-defensins were 0.6 –1.5 mol/L. At high concentrations, defensins (HNP-1 >2 mol/L) caused high-molecular-mass aggregates, comparable to Bacillus anthracis protective antigen and lethal factor. Conclusions: Our data indicate that toxin B interacts with high affinity with ␣-defensins and suggest that defensins may provide a defense mechanism against some types of clostridial glucosylating cytotoxins.
C
lostridium difficile is a major cause of antibioticassociated diarrhea and pseudomembranous colitis.1–3 The main pathogenicity factors implicated in these diseases are the exotoxins toxin A and toxin B produced by the pathogen. After uptake, autocatalytic
processing, and translocation into target cells, the toxins mono-O-glucosylate and thereby inactivate lowmolecular-mass guanosine triphosphate– binding proteins of the Rho family. This leads to cytotoxic effects, including depolymerization of the actin cytoskeleton.4 – 6 In addition, infections with C difficile and exposure of colonic cells to the toxins initiate massive cellular responses, including neutrophil infiltration linked to the onset of inflammation with up-regulation and/or release of cytokines such as interleukin-8,7 interleukin-6,8 interleukin-1,9 leukotriene B4,10 and interferon gamma.11 The 2 major groups of human antimicrobial peptides, defensins and cathelicidins, play an important role in the innate immune response. They seem to be implicated in various gastrointestinal diseases (for review, see Wehkamp et al12,13). Human defensins are cationic, amphipathic peptides of 3.5– 6 kilodaltons that are characterized by 3 intramolecular disulfide bonds. They are subdivided into ␣- and -defensins. Until now, 4 ␣-defensins were isolated from neutrophils (human neutrophil protein [HNP]-1 to HNP-4). Additionally, 2 enteric human ␣-defensins (HD-5 and HD-6) are found in the granules of Paneth cells of the small intestine (for review, see De and Contreras14). The microbicidal potential of ␣-defensins is mainly based on the insertion of the amphipathic peptides into bacterial membranes and damage of membrane integrity.15 Next to this bactericidal action, the peptides have signaling effects and influence inflammation, proliferation, wound healing, release of cytokines, and chemotaxis (for review, see Kim and Kaufmann16 and Shi17). Recently, HNPs were described to neutralize bacterial exotoxins. Kim et al reported that HNP-1–3 inhibit lethal factor (LF) from Bacillus anthracis. HNP-1 inhibits the protease activity of the toxin, and injection of HNP-1–3 protected mice from the intoxication by LF.18 The same group showed that HNP-1 neutralizes mono-ADP-ribosylating toxins (eg, Pseudomonas aeruginosa exotoxin A and diphtheria toxin) in vitro and in vivo.19 Abbreviations used in this paper: GTPase, guanosine triphosphatase; hBD, human -defensin; HD, human defensin; HNP, human neutrophil protein; IC50, median inhibitory concentration; LF, lethal factor; LT, lethal toxin; SDS-PAGE, sodium dodecyl sulfate/polyacrylamide gel electrophoresis; TER, transepithelial resistance. © 2008 by the AGA Institute 0016-5085/08/$34.00 doi:10.1053/j.gastro.2008.03.008
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Institut für Experimentelle and Klinische Pharmakologie und Toxikologie, Universität Freiburg, Freiburg, Germany
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These findings with exotoxins prompted us to test whether ␣-defensins also neutralize clostridial glucosylating toxins. Here, we present evidence that ␣-defensins inhibit the catalytic activity and cytotoxicity of the most potent cytotoxic factor of C difficile, toxin B.
Materials and Methods Materials, Antibodies, and Proteins
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Recombinant human ␣-defensin HNP-1, HNP-3, enteric ␣-defensin HD-5, and human -defensin (hBD)-1 were from Peptides International, Inc (Louisville, KY). A mix of human native HNP-1–3 and cathelicidin LL-37 were from Panatecs GmbH (Tübingen, Germany). Uridine 5=-diphosphate (UDP)-[14C]glucose (287.4 mCi/ mmol) was from PerkinElmer (Rodau-Jügesheim, Germany). All chemicals used in this study were of analytical grade and purchased from commercial sources. Anti-Rac1 monoclonal antibody clone 23A8, reacting with glucosylated and nonglucosylated Rac1, was from Upstate (Millipore Corp, Schwalbach, Germany); antiRac1 monoclonal antibody clone 102 recognizing only nonglucosylated Rac1 was from BD Biosciences Pharmingen (Heidelberg, Germany). Native toxins A and B from C difficile VPI 10463 and native lethal toxin (LT) from Clostridium sordellii 6018 were purified as described.20 Toxin A was additionally purified by thyroglobulin affinity chromatography.21 The catalytic domain of toxin B (amino acids 1–546) was expressed as glutathione S-transferase fusion proteins from the Escherichia coli expression vector pGEX-2T (Amersham Biosciences, Freiburg, Germany) as reported previously.22 The recombinant guanosine triphosphate– binding proteins RhoA, Rac1, and Cdc42 were prepared as glutathione S-transferase fusion proteins as described.23 The Bacillus anthracis toxins LF and protective antigen were a kind gift from R. J. Collier (Boston Medical School, Boston, MA).
Cell Culture and In Vivo Intoxication Experiments Swiss 3T3 and CaCo-2 cells were cultivated in Dulbecco’s modified Eagle medium at 37°C and 5% CO2 with medium containing 10% heat-inactivated fetal calf serum, 2 mmol/L L-glutamate, 100 U/mL penicillin, and 100 g/mL streptomycin and were trypsinized and reseeded 2 or 3 times a week. For all in vivo intoxication assays, the corresponding cells were washed twice with and kept in fetal calf serum–free Dulbecco’s modified Eagle medium. Antimicrobial peptides (1 and/or 3 mol/L, as indicated) were applied to the cells before toxin A or toxin B (5–100 pmol/L) was added. Untreated cells, either defensin alone or toxin A and toxin B alone, served as controls, respectively. For microscopic studies, Swiss 3T3 cells were grown in 24-well dishes and intoxicated with 50 pmol/L toxin A or 5 and 50 pmol/L toxin B after pretreatment with de-
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fensins (5 minutes or 3 hours, respectively). Pictures were taken with an AxioCam HRC camera and Axio Vision software (Carl Zeiss AG, Oberkochen, Germany). For the transepithelial resistance (TER) assay, CaCo-2 cells were seeded on 12-well Millicell cell culture inserts (Millipore Corp) and incubated 4 – 6 days with medium exchange every 2 days. Assays were performed when TER values reached ⬃1000 to 2000 ⍀/cm2. TER was determined with Endohm-12 (World Precision Instruments, Sarasota, FL). Defensins were preincubated with the cells for 2.5 hours before 100 pmol/L toxin A or toxin B was applied to the upper compartment of the Millicell cell culture inserts, and decrease of TER was monitored for up to 8 hours. For the detection of the glucosylation of Rac1 after preincubation with defensins (for 2.5 hours) and intoxication with 50 pmol/L holotoxin B, Swiss 3T3 cells grown in 24-well dishes were used. Sixty minutes after toxin application, the cells were washed with phosphate-buffered saline and lysed with Laemmli buffer, followed by Western blotting. Rac1 was detected using monoclonal antibodies clone 23A8 (1:5000) and clone 102 (1:10,000), followed by an anti-mouse immunoglobulin G horseradish peroxidase secondary antibody (1:5000; Biotrend, Cologne, Germany). Corresponding bands were detected using enhanced chemoluminescence.
In Vitro Glucosylation Experiments For initial studies, 9 nmol/L toxin B and 3 mol/L Rac1 were incubated at 37°C in the presence of 10 mol/L UDP-[14C]glucose in IVG buffer containing 50 mmol/L HEPES (pH 7.5), 100 mmol/L KCl, 2 mmol/L MgCl2, and 1 mmol/L MnCl2. After 2 minutes, defensins were added to a final concentration of 1 or 3 mol/L. Samples of 20 L were taken at indicated time points. Km values were determined with 1 nmol/L toxin B1–546, 1 mol/L Rac1, defensins HNP-1 and HNP-3 (final concentration, 1 mol/L), or HD-5 (3 mol/L) and varying UDP-[14C]glucose concentrations (0.5–20 mol/L). The reactions were incubated for 10 minutes at 37°C in IVG buffer. For determination of the median inhibitory concentration (IC50) values, 3 nmol/L toxin B or toxin B1–546 were preincubated with HNP-1 or HD-5 (0.1–5 mol/L) for 10 minutes at 37°C in the presence of 10 mol/L UDP[14C]glucose in IVG buffer supplemented with 100 g/mL bovine serum albumin. Glucosylation reaction was started by addition of 1 mol/L recombinant Rac1, RhoA, or Cdc42, respectively, and incubated for 15 minutes at 37°C. Total volume was 20 L. End point inhibition and reversibility of inhibition were examined with 3 nmol/L toxin B1–546, 1 mol/L recombinant or native defensins, and 5.5 mol/L Rac1. Toxin and antimicrobial peptides were preincubated for 15 minutes at 37°C in IVG buffer in a total volume of 22.5 L. The solution was split into 2 fractions (20 and 2.5 L) and the volume adjusted to 25 L each by
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addition of UDP-[14C]glucose (10 mol/L) and Rac1 (5.5 mol/L). Reactions were incubated for 10, 30, or 100 minutes at 37°C. UDP-[14C]glucose–labeled proteins were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) followed by phosphoimaging and densitometry. Quantification of signals and determination of IC50 and Km values were performed with ImageQuant (Amersham Biosciences) and SigmaPlot/Enzyme Kinetics module (Systat Software, Erkrath, Germany), respectively.
UDP-Sugar Hydrolase Assay
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UDP-sugar hydrolysis was essentially performed as described.23 Toxin B1–546 (100 nmol/L) pretreated with HNP-1 or HD-5 (0 –10 mol/L; 10 minutes, 37°C) was incubated with 20 mol/L UDP-[14C]glucose and 60 mol/L unlabeled UDP-glucose in a buffer containing 50 mmol/L HEPES (pH 7.5), 100 mmol/L KCl, 2 mmol/L MgCl2, 100 mol/L bovine serum albumin, and 100 mol/L MnCl2 for 30 minutes at 30°C. Samples were subjected to thin-layer chromatography with polyethyleneimine-cellulose plates (Merck, Darmstadt, Germany) and 0.2 mmol/L LiCl to separate the hydrolyzed sugar from UDP-sugars. The plates were dried and analyzed by Phosphorimager analysis (GE Healthcare, München, Germany). Quantification was performed with ImageQuant.
Aggregation Studies For turbidity assays, 3 g of each toxin (as indicated) were diluted in 500 L IVG buffer and incubated for 100 seconds before varying concentrations (0.5–3 mol/L) of the indicated peptides were injected in a volume of 50 L IVG buffer. Formation of aggregates was monitored by measurement of the increasing absorbance (light scattering) at 600 nm over time (up to 20 minutes) with a PerkinElmer LS 50 B luminescence spectrometer. Precipitation studies were performed either with 1.1 g toxin B1–546 or 2–3 g toxin B, B anthracis LF, B anthracis protective antigen, and C sordellii LT in a total volume of 25 L. Before addition of defensins, the protein solutions were centrifuged for 15 minutes at 13,000g and 4°C to segregate possible preformed protein complexes. The supernatants were incubated at rising concentrations of HNP-1 or HD-5 (0 –20 mol/L) for 10 minutes at 37°C. Following centrifugation (4°C, 45 minutes, 13,000g), supernatants and pellets were separated and transferred to SDS-PAGE. Proteins were visualized by Coomassie blue staining of the gels.
Results Cytotoxicity of Toxin B, but Not of Toxin A, Is Diminished by ␣-Defensins To test whether defensins and/or cathelicidin protect cells against the cytotoxic effects of clostridial glu-
Figure 1. ␣-Defensins neutralize toxin B, but not toxin A, in cell culture. Swiss 3T3 cells were preincubated with 3 mol/L HNP-1 or HD-5 in fetal calf serum–free medium. After 3 hours, the cells were intoxicated with 50 pmol/L toxin B or toxin A as indicated. Cell rounding was monitored by light microscopy and pictures were taken after 1 hour (toxin B) or 5 hours (toxin A). Corresponding controls as indicated.
cosylating toxins, we applied a mix of native HNP-1–3, recombinant HNP-1, HNP-3, HD-5, hBD-1, and cathelicidin LL-37 (3 mol/L each) to Swiss 3T3 cells before toxin application. The intoxication process characterized by cell rounding was monitored by light microscopy (exemplarily shown for HNP-1 and HD-5 in Figure 1). For toxin B, but not for toxin A, we found a strong reduction in cytotoxicity when the cells were pretreated with 3 mol/L of the corresponding ␣-defensins. In contrast, neither hBD-1 nor LL-37 protected cells from toxin B–induced cell rounding (not shown). HNPs were more effective than HD-5, which conferred only partial protection at a concentration of 3 mol/L. At 1 mol/L, the defensins did not protect or only weakly protected the cells against toxin B (not shown). The protective effect of HNP-1 persisted for up to 24 hours. Results were repro-
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duced with several cell lines (eg, HeLa, CaCo-2, HT-29, and Vero cells), excluding a cell type– dependent effect (not shown). To quantify reduction in cytotoxicity, the toxin-induced decrease in TER of human intestinal cells grown in a monolayer (CaCo-2 cells) was measured. As shown in Figure 2A, toxin B treatment from the apical site of the
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cell monolayer leads to a fast and strong reduction of the TER, with complete intoxication reached after about 4 –5 hours. HNP-1 3 mol/L strongly delayed intoxication with toxin B. HNP-1 1 mol/L displayed a weak protective effect (Figure 2A). Again, HD-5 was less effective even at a final concentration of 3 mol/L (Figure 2B). For toxin A, we found no significant influence of either defensin on the cytotoxic potential (Figure 2C), corroborating the microscopic studies. An Rac1 antibody that recognizes only nonglucosylated Rac124,25 was used to monitor the glucosylation status of Rac1 after intoxication of cells pretreated with the corresponding defensins (as described previously). HNP-1 and HD-5 inhibited toxin B–induced glucosylation of Rac1 in a concentration-dependent manner (Figure 2D). Again, HNP-1 displayed a stronger effect than HD-5 with complete inhibition of glucosylation at a final concentration of 3 mol/L. The same concentration of HD-5 reduced the glucosylation only to about 30%.
␣-Defensins Inhibit Toxin B–Mediated Glucosylation of Small Guanosine Triphosphatases
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The inhibiting effects of ␣-defensins on glucosyltransferase activity of toxin B were studied in an in vitro glucosylation assay. First, reactions with toxin A and toxin B were started without defensins. HNP-1, HNP-3, or HD-5 (1 and 3 mol/L each) were added after 2 minutes to the reaction mixture. In case of toxin B, HNP-1 (Figure 3A) and HNP-3 (Figure 3B) led to an immediate and concentrationdependent decrease of the glucosyltransferase activity with strong or even complete inhibition at 3 mol/L. Inhibition by HD-5 was delayed with an onset of a decline after 5 minutes (at 3 mol/L) or 10 minutes (at 1 mol/L, Figure 3C), respectively. A mix of native HNPs (HNP-1–3) exhibited strong inhibition of glucosyltransferase activity comparable to recombinant HNP-1 and HNP-3 (Supplementary Figure 1A; see supplementary material online at www.gastrojournal. org). Glucosyltransferase activity of toxin A was not influenced by addition of HNP-1 (Supplementary Figure 1B; see supplementary material online at www.gastrojournal.org) or a mix of native HNP-1–3 (not shown).
4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™
Figure 2. ␣-Defensins delay toxin B–mediated intoxication in vivo by inhibition of glucosylation of small GTPases. (A–C) Human intestinal epithelial cells (CaCo-2) grown on Millicell filter inserts were preincubated with 1 and 3 mol/L HNP-1 or HD-5 for 2.5 hours before toxin A or toxin B (100 pmol/L each) was added (as indicated). Decrease of TER was monitored at indicated time points. Starting resistance was set to 100%, and TER values are given as percent of starting resistance. “Control,” untreated cells. (D) Swiss 3T3 cells were preincubated with indicated ␣-defensins and intoxicated with 50 pmol/L toxin B as described previously. After onset of cell rounding (60 minutes), cells were lysed and transferred to SDS-PAGE and Western blotting. Rac1 was detected with 2 different monoclonal anti-Rac1 antibodies, one recognizing unmodified and glucosylated Rac1 (“total Rac1,” input control) and the second recognizing only unmodified Rac1 (“non-Glc Rac1”). Controls without toxin as indicated.
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The corresponding Km values for HNP-1, HNP-3, and HD-5 were determined using the catalytic domain of toxin B encompassing amino acids 1–546 (toxin B1–546), Rac1, and varying UDP-[14C]glucose concentrations (0.5–20 mol/L). Defensins and UDP-glucose were added simultaneously to the reaction. Control glucosylation reaction without defensin resulted in a Km value of 2.4 mol/L. Addition of ␣-defensins inhibited glucosyltransferase reaction in a competitive manner, with HNP-1 and HNP-3 elevating corresponding Km values about 10-fold to 23.8 mol/L (HNP-1, Figure 3D) and about 9-fold to 21.1 mol/L (HNP-3, Figure 3E), respectively. At 1 mol/L, HD-5 elevated Km only by the factor 2 (not shown). When the concentration of HD-5 was raised to 3 mol/L, the Km value increased about 6- to 7-fold (Figure 3F).
Reversibility of End Point Inhibition Next to recombinant and native human ␣-defensins, hBD-1 and cathelicidin LL-37 were tested for their inhibitory potential. To guarantee maximal inhibition, the defensins and toxin B1–546 were preincubated for 15 minutes at 37°C before Rac1 (5.5 mol/L) and UDP-
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Figure 3. Characterization of inhibition of toxin B by ␣-defensins in vitro. (A–C) Glucosylation reactions were started without defensins in the presence of 5.5 mol/L Rac1 and 9 nmol/L toxin B. (A) HNP-1, (B) HNP-3, or (C) HD-5 were added after 2 minutes in a final concentration of 1 or 3 mol/L, respectively. Samples were taken at the indicated time points, and the amount of [14C]-labeled Rac1 was analyzed by SDS-PAGE, autoradiography, and densitometry. Signal intensity of samples without defensin at t ⫽ 10 or 25 minutes was set to 100%, and relative glucosyltransferase activity of each sample is shown as percent glucosylation of the corresponding control (n ⱖ 3 ⫾ SEM). (D–F) Km values of samples without defensin (“control”), with (D) 1 mol/L HNP-1, (E) 1 mol/L HNP-3, or (F) 3 mol/L HD-5 were determined at varying UDP-[14C]glucose concentrations (0.5–20 mol/L) in the presence of 1 mol/L Rac1 and 1 nmol/L toxin B1–546. Corresponding Michaelis–Menten kinetics (n ⱖ 3 ⫾ SD) and respective Km values are shown. Glucosyltransferase activity (rate [1/s]) of toxin B1–546 is given as pmol glucosylated Rac1⫺1 · pmol enzyme⫺1 · s.
[14C]glucose (10 mol/L) were added. These conditions were found to enhance defensin-mediated inhibition (see Supplementary Figure 2; see supplementary material online at www.gastrojournal.org). Neither hBD-1 nor LL-37 reduced glucosyltransferase activity of toxin B (Figure 4A), corroborating the in vivo data from cell intoxication. Reversibility of defensin-induced inhibition was assessed by diluting the preincubated defensin/toxin mix by a factor of 10, resulting in a final concentration of 0.1 mol/L defensin and 0.3 nmol/L toxin B1–546 at constant Rac1 and UDP-[14C]glucose concentrations. This dilution partially reconstituted glucosyltransferase activity, indicating reversibility of defensin-mediated inhibition. To visualize corresponding signals in a comparable manner, undiluted samples were incubated for 10 minutes and diluted samples for 100 minutes (Figure 4A). For statistical analysis (Figure 4B), all probes were incubated for 30 minutes and signals normalized against corresponding controls. Partial reversibility of the inhibiting effects suggested that the overall structure of the toxin was not changed to a large extent by the defensins. This view
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was supported by the finding that the sensitivity of the toxin toward V8-protease was not strongly affected by the presence of HNPs (Supplementary Figure 3; see supplementary material online at www.gastrojournal .org). Partial protection of cleavage observed with HD-5 may be based on the strong aggregation of toxin B (see following text) and reduced accessibility of cleavage sites. To determine whether the 3-dimensional structure of defensins is crucial for their inhibitory potential, HNP-1 and HD-5 were incubated with the reducing agent dithiothreitol or heated at 95°C for 5 minutes. The corresponding defensins were then applied in the in vitro glucosylation assay as described previously. Treatment with dithiothreitol (10 mmol/L) largely abolished the inhibitory potential of HNP-1 and HD-5 (Figure 4C), indicating that an intact overall structure stabilized by internal disulfide bonds is essential for the inhibition. Heating of the peptides to 95°C did not influence the inhibitory potential.
Determination of IC50 Values
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Figure 4. End point inhibition: specificity, reversibility, and sensitivity toward dithiothreitol. (A and B) To guarantee maximal inhibition by the antimicrobial peptides, toxin B1–546 (3 nmol/L) was preincubated for 15 minutes at 37°C with indicated defensins or cathelicidin LL-37 (1 mol/L each). Part of the sample was diluted 1:10 before Rac1 (5.5 mol/L) and UDP[14C]glucose (10 mol/L) were added to start glucosylation. Samples were incubated for 10 minutes (“undiluted”), 100 minutes (“diluted”), or 30 minutes (B). Radioactive labeled Rac1 was detected by SDS-PAGE and autoradiography. (A) Reappearing signals indicate partial reversibility of the inhibition. (B) Statistical analysis of signal intensities normalized against corresponding controls (n ⫽ 3 ⫾ SEM). (C) Toxin B1–546 (3 nmol/L) was preincubated with HNP-1 or HD-5 (3 mol/L each) ⫾ 10 mmol/L dithiothreitol in a total volume of 20 L for 10 minutes at 37°C. Heating of defensins (95°C, 5 minutes) was performed before preincubation with the toxin. Toxin B1–546 pretreated only with reducing agents served as control. Glucosylation reaction was initiated by addition of 1 mol/L Rac1. Samples were taken after 15 minutes, and radioactively labeled Rac1 was detected by SDS-PAGE and autoradiography.
The concentration-dependent effect of HNP-1 and HD-5 on the glucosylation of the main substrates of toxin B, namely RhoA, Rac1, and Cdc42 (1 mol/L each), was tested by the in vitro glucosylation assay. To guarantee maximal inhibition, a preincubation step of 10 minutes (as described previously) was applied. In all cases, treatment of the toxins with either HNP-1 or HD-5 strongly reduced the glucosylation of the low-molecularmass guanosine triphosphatases (GTPases) with IC50 values ranging from 0.6 to 1.5 mol/L, estimated from the corresponding regression curves (exemplarily represented in Figure 5A and summarized in Table 1). As expected, HNP-1 displayed slightly lower IC50 values compared with HD-5. Based on the preincubation step, which counteracts the delayed onset of inhibition by HD-5 (see Figure 3C), the differences between HNP-1 and HD-5 are not as pronounced as seen for the corresponding Km values. Next to the inhibition of glucosyltransferase activity, ␣-defensins also inhibited glucohydrolase activity of toxin B1–546 (HNP-1: IC50 ⫽ 3.8 mol/L; HD-5: IC50 ⫽ 6.8 mol/L; see Figure 5B), corroborating a direct effect of the defensins on the catalytic activity of the toxin.
Toxin Aggregation Induced by Defensins Under various experimental settings utilizing high defensin concentrations (eg, for gel filtration analysis), we observed that defensins induced aggregation of toxin B. Apparently these aggregates were of high molecular mass because they were precipitated at low g-force (13,000g). To determine the concentrations of ␣-defensin, which caused complex formation and/or precipitation of toxin B, we performed a precipitation
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assay after preincubation of toxin B with rising concentrations of HNP-1. In case of toxin B1–546, which proved to be most sensitive to formation of aggregates (Supplementary Figure 4; see supplementary material online at www.gastrojournal.org), precipitation started at ⬃2 mol/L HNP-1 (Figure 6A). As shown in Figure 6A, HNP-1 was shifted to the pellet fraction together with the toxin. Estimated from Coomassie blue– stained gels, approximately 18 pmol toxin B1–546 coprecipitated up to ⬃250 pmol HNP-1, indicating multiple binding sites for HNP-1. Comparable precipitation studies were performed with toxin A, toxin B, LT, and B anthracis LF and protective antigen in combination with HNP-1 and HD-5 (Supplementary Figure 4; see supplementary material online at www.gastrojournal .org). Except LT and toxin A, which were not inhibited by the corresponding defensins, all toxins tested formed aggregates. HD-5 precipitated toxins more efficiently than HNP-1. Aggregate formation was further characterized by turbidity assays. Again, HD-5 proved to be most potent in aggregate formation in combination with toxin B1–546, followed by HNP-1 and HNP-3 (Figure 6B). Neither hBD-1 nor cathelicidin LL-37 induced any detectable aggregation, excluding a mere charge effect. Although full-length toxin B is much larger in size than the isolated catalytic domain, it proved to aggregate to the same extent or even less. Accordingly, aggregation was mainly mediated by the N-terminal part of toxin B (amino acids 1–546). B anthracis LF and protective antigen also formed aggregates under the same conditions (Supplementary Figure 5; see supplementary material online at www.gastrojournal.org).
Discussion Here we report that the ␣-defensins HNP-1, HNP-3, and enteric HD-5 prevent the cytotoxic effects of
Table 1. IC50 Values Figure 5. Determination of the IC50 values for glucosyltransferase and glucohydrolase activity. (A) Glucosyltransferase activity. Toxin B (3 nmol/L) was preincubated with rising concentrations (0.1–5 mol/L) of HNP-1 or HD-5 in a total volume of 20 L for 10 minutes at 37°C. Glucosylation reaction was started by adding 1 mol/L Rac1. Radioactively labeled Rac1 was detected by SDS-PAGE and autoradiography followed by densitometry for statistical evaluation. Total Rac1 (input control) is shown by Coomassie blue–stained gels (upper panels, as indicated). (B) Glucohydrolase activity. Toxin B1–546 (100 nmol/L) was preincubated with rising concentrations (0 –10 mol/L) of HNP-1 or HD-5 in a total volume of 10 L for 10 minutes at 37°C. Glucohydrolase reaction was started by the addition of 20 mol/L UDP-[14C]glucose. Incubation was for 30 minutes at 30°C. UDP-[14C]glucose hydrolyzed was analyzed by thin-layer chromatography, autoradiography, and densitometry. (A and B) Signal intensity of samples without defensin was set to 1.0, and relative glucosyltransferase/glucohydrolase activity of each sample is shown (n ⱖ 3 ⫾ SD). IC50 values for the main substrates of toxin B were determined from the corresponding regression curves (see Table 1).
Toxin (3 nmol/L) Toxin B
GTPase (1 mol/L) Rac1 RhoA Cdc42
Toxin B1–546
Rac1 RhoA Cdc42
Defensin (0–5 mol/L)
IC50 value (mol/L)
HNP-1 HD-5 HNP-1 HD-5 HNP-1 HD-5 HNP-1 HD-5 HNP-1 HD-5 HNP-1 HD-5
0.6 0.8 0.8 1.1 0.7 0.6 0.6 1.2 1.0 1.5 0.7 1.5
NOTE. Listed are all determined combinations of toxin B and toxin B1–546 with the main protein substrates Rac1, RhoA, and Cdc42, respectively. IC50 values were determined as described in Figure 5.
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Figure 6. Toxin aggregation induced by ␣-defensins. (A) Toxin B1–546 was preincubated for 15 minutes at 37°C with rising concentrations of HNP-1 (0 –20 mol/L). Samples were centrifuged at 13,000g at 4°C for 50 minutes. Supernatants were separated from pellets and the individual fractions transferred to SDS-PAGE. Coomassie blue–stained gels exhibiting the toxin protein (63 kilodaltons) and defensin (3.5 kilodaltons) bands are shown. (B) A solution containing 3 g toxin B1–546 was incubated for 100 seconds before 3 mol/L (final concentration) of the indicated antimicrobial peptides was injected. Aggregate formation in solution was monitored by increase in turbidity at OD600 over time. Intensity (Int) of light scattering is given as arbitrary units.
toxin B in intestinal epithelial cells and in a large array of other cell types. The ␣-defensins inhibited the glucosylation of Rho proteins by toxin B, which is suggested to be the molecular basis for the cytotoxic effects of the toxin. Because the defensins blocked the glucohydrolase activity of toxin B and did not affect the GTPase activities of Rho proteins per se (not shown), our data indicate that the inhibiting effect is caused by blockade of the catalytic activity of the toxin. This view is supported by the finding
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that ␣-defensins compete with the binding of UDP-glucose to the glucosyltransferase domain of toxin B. The substrate competition at the active site is characterized by an up to 10-fold increase in the Km value for UDPglucose. The inhibition is specific for toxin B, because toxin A is not influenced by the defensins under the same conditions. The effect is specific for ␣-defensins, because hBD-1 and cathelicidin LL-37 did not influence glucosyltransferase activity. The dilution experiments suggest that inhibition of glucosyltransferase activity by defensins is reversible at least at low concentrations of the inhibitors, which is in agreement with the Michaelis– Menten kinetics obtained. The unaltered sensitivity of HNP-treated toxin B toward proteolytic cleavage by V8protease (Supplementary Figure 3; see supplementary material online at www.gastrojournal.org) strongly suggests that the binding of the ␣-defensins to toxin B does not cause major changes in the toxin structure and does not merely result in a denaturating effect. Regarding the concentration-dependent inhibition, we obtained IC50 values at a low range (0.6 –1.5 mol/L) for toxin B and HNP-1 or HD-5. Defensins occur at very high concentrations in specific intestinal parts. Recently, it has been estimated that 0.5 mg of HD-5 is stored per square centimeter of ileal mucosa with concentrations of 50 – 250 g/mL (⬃14 to 70 mol/L) in the intestinal lumen.26 Thus, this concentration would largely block the action of toxin B, suggesting a protective role of defensins toward the potent effects of C difficile toxin B. HD-5 is produced by Paneth cells, which are usually in the small intestine but not in the colon, and only metaplastic Paneth cells are observed in the inflamed colon. However, C difficile is also capable of colonizing the small intestine, where it may act as an asymptomatic reservoir for disease.27 Whether the low virulence of C difficile in the small intestine is related to the occurrence of toxininactivating peptides remains to be clarified. Our findings add another exotoxin to the list of bacterial toxins, which were reported to be inhibited by defensins. Among these are the LF from B anthracis, diphtheria toxin, and Pseudomonas exotoxin A. Interestingly, B anthracis LF (IC50 ⫽ ⬃0.2 mol/L) and the mono-ADP-ribosyltransferases diphtheria toxin (IC50 ⫽ ⬃11.1 mol/L) and exotoxin A (IC50 ⫽ ⬃19.2 mol/L) are inhibited at similar concentrations of defensins as observed for toxin B.18,19 Also, the small concentration range observed for inhibition of toxin B with a weak protection at 1 mol/L and almost complete protection at 3 mol/L is in line with the action of HNP-1 on anthrax LT.18 Thus, the interaction of defensins with these toxins might be based on related mechanisms. The direct effect of defensins on catalytic activity is a plausible cause for their inhibiting potential. However, we cannot exclude additional effects on the receptor and/or uptake of toxin B in vivo. We observed a concentration-dependent aggregation of toxin B in the presence of ␣-defensins. Notably, the effi-
ciency of ␣-defensins to induce aggregation of toxin B is not paralleled by inhibition of glucosyltransferase activity. HD-5 induced formation of aggregates more efficiently than HNP-1, but it was less efficient than HNP-1 in inhibiting glucosyltransferase activity. Also, HNPs inactivated glucosyltransferase activity of holotoxin B immediately, whereas aggregation started with a significant delay. Regarding the in vivo situation, it is noteworthy that aggregation of holotoxin B by HNP-1 started at defensin concentrations higher than the corresponding IC50 values. Therefore, aggregate formation is, at least in part, independent of the inhibitory effects of HNPs. Neither toxin A nor LL-37 nor hBD-1 displayed aggregate formation. In the case of HD-5, we cannot rule out an influence of strong aggregate-forming potential on the observed inhibitory activity. The complex formation seems to be a common feature, because B anthracis LF and protective antigen, which were used as control proteins in this context, displayed the same behavior. Therefore, we believe that this observation may represent an additional mode of action for specific defensins (eg, HD-5) or at sites where high defensin concentrations are present, for example, within specialized regions of the small intestine or phagocytic vacuoles of neutrophils. We were surprised to find that in contrast to toxin B, toxin A was not significantly inactivated by HNP-1 under the in vivo conditions tested. The same was true for C sordellii LT, which is even more similar in structure to toxin B than toxin A. These different effects observed with toxin B and toxin A or LT indicate the specificity of the interaction of the defensins with toxin B. Early studies on C difficile infections and pathogenicity referred to “enterotoxin A” as the causative agent for pseudomembranous colitis and antibiotic-associated diarrhea.28 Based mainly on the frequent isolation of toxin A–negative, toxin B–positive strains from patients with C difficile–associated diseases, the potent “cytotoxin B” seems to be the responsible pathogenic agent in many cases.29 –32 In this respect, the selectivity of human ␣-defensins HNP-1, HNP-3, and HD-5 for toxin B is remarkable. It remains to be clarified whether a high-affinity interaction of toxin B with defensins is at least in part responsible for the former characterization of toxin B as a cytotoxin without enterotoxic activity, which was deduced from various experimental animal colitis models. Taken together, we observed that defensins interact with high affinity with C difficile toxin B to inhibit its activity. This finding offers a new perspective in host-pathogen interaction in C difficile infections and associated diseases.
Supplementary Data Note: To access the supplementary material accompanying this article, visit the online version of Gastroenterology at www.gastrojournal.org, and at doi:10.1053/j.gastro. 2008.03.008.
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31. van den Berg RJ, Claas EC, Oyib DH, et al. Characterization of toxin A-negative, toxin B-positive Clostridium difficile isolates from outbreaks in different countries by amplified fragment length polymorphism and PCR ribotyping. J Clin Microbiol 2004; 42:1035–1041. 32. Samra Z, Talmor S, Bahar J. High prevalence of toxin A-negative toxin B-positive Clostridium difficile in hospitalized patients with gastrointestinal disease. Diagn Microbiol Infect Dis 2002;43: 189 –192.
Received October 16, 2007. Accepted March 6, 2008. Address requests for reprints to: Klaus Aktories, Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Albertstrasse 25, D-79104 Freiburg, Germany. e-mail: klaus.aktories@pharmakol. uni-freiburg.de; fax: (49) 761-2035311. Supported by the Deutsche Forschungsgemeinschaft. The authors report that no conflicts of interest exist.
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