N-Chlorotaurine Inacitvates Shiga Toxin Figure 5. Impact of NCT on purified Stx2 demonstrated in gel electrophoresis. (A) Purified Stx2 was treated with 55 mM NCT for 30 min at RT. Then, an aliquot was used for SDS-PAGE. Reducing buffer contained 4.5% v/v mercaptoethanol. A gel containing bands of aliquots containing 10 or 15 mg Stx2 is shown (one representative of three independent experiments). Bands a–f were subjected to in-gel digestion and mass spectrometry. (B) Purified Stx2 was treated with 55 mM NCT for 30 min at RT and subjected to a tricine gel for improved separation of the low molecular weight bands. Note the separation of the B subunit in bands a and b, which were subjected to in-gel digestion and mass spectrometry. doi:10.1371/journal.pone.0047105.g005 Materials and Methods Chemicals, Test Solutions, and Media Pure NCT as a crystalline sodium salt (mol wt 181.57 g/mol) was prepared according to [11]. Purity was proved by spectro- photometry [11]. Pure NVC-612 (molecular weight 209.63 g/ mol) and NVC-422 (molecular weight 244.07 g/mol) were from NovaBay Pharmaceuticals Inc. (Emeryville, CA, USA). Fresh solutions of 55 mM (equals 1% w/v NCT) or 550 mM (equals 10% w/v NCT) of each of these test substances were prepared in 0.1 M phosphate buffer at pH 7.1 and diluted in this buffer to the final concentrations as appropriate. Mueller-Hinton agar was from Merck, fetal calf serum from Invitrogen (10% v/v in RPMI), RPMI-1640 Medium from Sigma and EHEC Direct Medium 1055 rp from Heipha (Heipha Dr. M u¨ller GmbH, Eppelheim, Germany). The EHEC Direct Medium was used to grow EHEC. In contrast to conventional media, it contains 50 ng/ml mitomy- cin C, which stimulates the Stx production. EBM-2 medium with growth supplemental kit was from LONZA Cologne AG (Ko¨ln, Germany). Purified Stx2 (5 mg/ml), a kind gift from Helge Karch (Mu¨ nster, Germany), was used to perform gel electrophoresis and mass spectrometry, FACS analysis and confocal microscopy [38]. Growth of EHEC and Stx2 Production in the Presence of Sublethal Concentrations of NCT Clinical isolates EHEC 178 and EHEC 22 (both O157:H7, Stx1 negative, positive for Stx2, eae-coding intimin, and serine protease EspP), all deep frozen for storage, were grown on Mueller-Hinton agar plates and subsequently in EHEC Direct Medium at an agitation of 160/min and 37uC overnight to 3–56108 colony forming units (CFU)/ml. Stock solutions of NCT and NVC-612 (55 mM each) were diluted to 16.5 mM, 22 mM, and 27.5 mM in 0.1 M phosphate buffer, NVC-422 to 5.5 mM, 11 mM, and 16.5 mM. Bacterial suspensions (40 ml) were mixed with 3560 ml EHEC Direct Medium and 400 ml of the prepared test solutions, respectively. After 0, 2, 4, and 6 h at 37uC, aliquots of 200 ml were removed, and the half (100 ml) from these was diluted 100-fold in 0.9% w/v NaCl. Immediately afterwards, aliquots (50 ml) of these dilutions were spread in duplicate on tryptic soy agar plates with an automatic spiral plater (model WASP 2, Don Whitley Scientific, Shipley, UK), allowing a detection limit of 100 CFU/ ml taking into account both plates and the dilution. Addition of inactivation substances for the used oxidants was not necessary under these test conditions as tested in a pilot experiment. The plates were incubated at 37uC, and colony forming units of microorganisms were counted after 24 and 48 h. To determine the presence of Stx2 in the samples, the other half of the aliquots (100 ml) of these dilutions were subjected to Stx Premier ELISA Kit (Meridian Bioscience, Inc. Cincinnati, OH, USA) in accor- dance to the instructions of the manufacturer. Influence of NCT on the Cytotoxic Effect of Stx2 against Vero Cells Vero cells (kidney monkey cells, ATCC CCL-81) were grown at 37uC and 5% v/v CO2 in RPMI 1640 medium plus 10% v/v fetal calf serum in cell culture flasks. Subcultures were grown in 96 well microtitre plates to a density of approximately 90% confluency. EHEC 178 and 22 were grown in EHEC Direct Medium overnight as described above. After centrifugation, the superna- tants were sterile filtered (0.45 mm, Sartorius). Solutions of 550 mM, 55 mM, 27.5 mM and 13.75 mM NCT, NVC-422, and NVC-612 each were prepared in 0.1 M phosphate puffer (pH 7.1). 900 ml of filtered supernatants were mixed with 100 ml of test solutions (NCT, NVC-422, or NVC-612) and incubated for 30 min. Additional experiments were done with 55 mM NCT and incubation times of 1, 5, 10, 15, 20, 25, and 30 min. Subsequently, samples were 100-fold diluted in RPMI + 10% v/v FCS to decrease the concentration of N-chloro amino acids under the toxic level for cells (toxic threshold.0.55 mM NCT in RPMI + 10% v/v FCS with unlimited incubation,.5.5 mM NCT in RPMI + 10% v/v FCS or even in non-chlorine-consuming buffers for at least 60 min incubation). 100 ml each of these diluted samples were given to the cells in 100 ml RPMI + FCS. After 72 h, cells that showed the typical cytopathic effect caused by Stx2 were counted at a 100-fold magnification. To confirm these results, cell viability was evaluated by trypan blue exclusion test and release of lactate dehydrogenase (Cytotoxicity Detection Kit LDH, Roche, Mannheim, Germany) in accordance to the instructions of the manufacturer. Controls without oxidants, without EHEC super- natant, with taurine or dimethyltaurine and supernatant, with plain RPMI + FCS, and with supernatant of E. coli ATCC 11229, a Shiga toxin negative strain, were performed in parallel. Igepal CA-630 1% (Sigma), which lyses the cells, was used as a positive control for the LDH assay. Consumption of Oxidation Capacity by EHEC Medium Since active chlorine compounds react with organic residues of media such as the used EHEC Direct Medium [11,12], oxidative activity is consumed in part (so-called chlorine consumption). This effect has to be taken into account for correct interpretation of the results, if the application of such media can’t be avoided. Therefore, we measured the decrease of oxidation capacity with redox-iodiometry [39]. To gain similar conditions as in the Vero cell assays, 3600 ml of EHEC Direct Medium were mixed with 400 ml of buffered NCT, NVC-422, or NVC-612 solutions to get final concentrations of 55 mM, 5.5 mM, 2.75 mM and 1.375 mM. Subsequently, the redox potential after reaction with iodide was determined using a PHM250 Ion Analyzer (Radiom- eter, Copenhagen, Denmark), and the remaining oxidation capacity was calculated as described previously [39]. Binding of NCT-treated Stx2 to GEnC (FACS Analysis) Human kidney glomerular endothelial cells (GEnC) were a kind gift from Simon Satchell PhD, Academic Renal Unit, University of Bristol [40]. GEnC were cultured in EBM-2 medium and growth factor kit as described [41]. Purified Stx2 was incubated with 55 mM NCT (1% w/v) in PBS at 37uC for 30 min. For FACS analysis, cells were incubated with 20 pg/ml NCT-treated Stx2. Controls with Stx2 in PBS or without Stx2 were performed in parallel. Cells were incubated for 4 h at 37uC. Stx2 bound on the cell surface was measured using a mouse-anti-Stx2-antibody purchased from Hycult biotech (Uden, Netherlands). Secondary antibody was FITC-labeled polyclonal goat-anti-mouse (DAKO, Glostrup, Denmark). Antibody dilutions were chosen as given in PLOS ONE | www.plosone.org 9 November 2012 | Volume 7 | Issue 11 | e47105