Human α-Defensins Inhibit Clostridium difficile Toxin B Torsten Giesemann, Gregor Guttenberg, Klaus Aktories  Gastroenterology  Volume 134, Issue 7, Pages 2049-2058 (June 2008) DOI: 10.1053/j.gastro.2008.03.008 Copyright © 2008 AGA Institute Terms and Conditions

Figure 1 α-Defensins neutralize toxin B, but not toxin A, in cell culture. Swiss 3T3 cells were preincubated with 3 μmol/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. Gastroenterology 2008 134, 2049-2058DOI: (10.1053/j.gastro.2008.03.008) Copyright © 2008 AGA Institute Terms and Conditions

Figure 2 α-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 μmol/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 α-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. Gastroenterology 2008 134, 2049-2058DOI: (10.1053/j.gastro.2008.03.008) Copyright © 2008 AGA Institute Terms and Conditions

Figure 3 Characterization of inhibition of toxin B by α-defensins in vitro. (A–C) Glucosylation reactions were started without defensins in the presence of 5.5 μmol/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 μmol/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 ≥ 3 ± SEM). (D–F) Km values of samples without defensin (“control”), with (D) 1 μmol/L HNP-1, (E) 1 μmol/L HNP-3, or (F) 3 μmol/L HD-5 were determined at varying UDP-[14C]glucose concentrations (0.5–20 μmol/L) in the presence of 1 μmol/L Rac1 and 1 nmol/L toxin B1–546. Corresponding Michaelis–Menten kinetics (n ≥ 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. Gastroenterology 2008 134, 2049-2058DOI: (10.1053/j.gastro.2008.03.008) Copyright © 2008 AGA Institute Terms and Conditions

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 μmol/L each). Part of the sample was diluted 1:10 before Rac1 (5.5 μmol/L) and UDP-[14C]glucose (10 μmol/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 μmol/L each) ± 10 mmol/L dithiothreitol in a total volume of 20 μL 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 μmol/L Rac1. Samples were taken after 15 minutes, and radioactively labeled Rac1 was detected by SDS-PAGE and autoradiography. Gastroenterology 2008 134, 2049-2058DOI: (10.1053/j.gastro.2008.03.008) Copyright © 2008 AGA Institute Terms and Conditions

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 μmol/L) of HNP-1 or HD-5 in a total volume of 20 μL for 10 minutes at 37°C. Glucosylation reaction was started by adding 1 μmol/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 μmol/L) of HNP-1 or HD-5 in a total volume of 10 μL for 10 minutes at 37°C. Glucohydrolase reaction was started by the addition of 20 μmol/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 ≥ 3 ± SD). IC50 values for the main substrates of toxin B were determined from the corresponding regression curves (see Table 1). Gastroenterology 2008 134, 2049-2058DOI: (10.1053/j.gastro.2008.03.008) Copyright © 2008 AGA Institute Terms and Conditions

Figure 6 Toxin aggregation induced by α-defensins. (A) Toxin B1–546 was preincubated for 15 minutes at 37°C with rising concentrations of HNP-1 (0–20 μmol/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 μg toxin B1–546 was incubated for 100 seconds before 3 μmol/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. Gastroenterology 2008 134, 2049-2058DOI: (10.1053/j.gastro.2008.03.008) Copyright © 2008 AGA Institute Terms and Conditions